Mutants of thymidylate synthase and uses thereof

ABSTRACT

The present invention provides a mutated human TS, said mutated synthase differing from wild type TS at amino acid residue 49, amino acid residue 52, amino acid residue 108, amino acid residue 221 or amino acid residue 225. Also provided is cDNA mutated human TSs and novel vectors and host cells and methods of using the mutated human TSs.

This is US national stage application of international applicationPCT/US98/02145 filed Feb. 3, 1998, which claims benefit of priorityunder 35 USC 119(e) of provisional U.S. application Ser. No. 60/037,163,filed Feb. 4, 1997, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of gene therapyand biochemical pharmacology. More specifically, the present inventionrelates to mutants of human enzyme thymidylate synthase and usesthereof.

2. Description of the Related Art

Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the rate limiting stepin the sole de novo biosynthesis pathway to thymidylate, which isnecessary for DNA synthesis and repair (Carreras et al., 1995). Themechanism of TS activity involves the reductive methylation of thesubstrate, 2′-deoxyuridine 5′-monophosphate (dUMP) by transfer of amethylene group from the cofactor, 5,10-methylenetetrahydrofolate(CH₂H₄folate), to generate 2′-deoxythymidine 5′-monophosphate (dTMP) and7,8-dihydrofolate (H₂folate). The inhibition of the TS pathway resultsin a thymineless state, which is toxic to rapidly dividing cells whichhave a high dTTP demand for DNA synthesis. This cytotoxicity is causedby DNA fragmentation and misincorporation of dUTP due to dTTP depletion.If there is enough supplied exogenous thymidine, cells survive throughthe salvage pathway depending on the use of thymidine kinase (TK).However, in normal tissues and in some tumor cells, the concentrationsof circulating thymidine may not be sufficient to keep cells normallygrowing (Touroutoglou et al., 1996).

As a consequence, TS is an attractive target for anti-cancer drug designdue to its crucial role in maintaining pools of thymidylate for DNAsynthesis. Since the 1950s, many analogues of both the pyrimidinesubstrate (dUMP) and folate cofactor (CH₂H₄folate) have been synthesizedand tested as potential anti-cancer therapeutics. However, although anumber of inhibitors that tightly bind to TS were discovered, before1995, 5-fluorouracil (5-FU) was the sole TS-targeted drug approved forclinical application. In vivo, 5-FU is metabolized to5-fluoro-2-deoxyuridylate (FdUMP) that subsequently occupies the dUMPbinding site forming a ternary complex with the enzyme and the folatecofactor, resulting in inhibition of TS. As the three-dimensionalstructures of TS have been revealed, the folate binding site in TS hasbeen explored for the design of highly specific inhibitors (Jackman etal., 1995b), and have led to the emergence of novel folate analogues,such as tomudex (ZD1694), BW1843U89, AG331 and AG337 etc. These agentsas the new generation TS-directed inhibitors have entered clinical trailin recent years. The approval of tomudex for treatment of advancedcolorectal cancer in the United Kingdom occurred last year.

The major blood folate is 5-methyl-tetrahydrofolate (5-CH₃—H₄folate),which enters cells via membrane transports [or called reduced folatecarriers (RFC)]. Once inside the cell, 5-CH₃—H₄folate is metabolized bymethionine synthase to tetrahydrofolate. This coenzyme is converted byserine hydroxymethyltransferase to 5,10-methylene-tetrahydrofolate andalso polyglutamated by folylpolyglutamate synthase (FPGS) to become5,10-methylene-tetrahydrofolate polyglutamates [CH₂H₄folate(Glu)_(n)].CH₂H₄folate(Glu)_(n), as a cofactor, donates its one-carbon unit and twoelectrons to the reductive methylation reaction converting dUMP to dTMP.Dihydrofolate (H₂folate) is a product of this process, which requiresthe sequential action of dihydrofolate reductase (DHFR) and serinehydroxymethyltransferase in order to resynthesize CH₂H₄folate(Glu)_(n).Inhibition of DHFR by methotrexate (MTX) may lead to an accumulation offolates in the inactive H₂folate form, resulting in depletion ofCH₂H₄folate and dTMP.

There is much interest in correlating enzyme structure and functionusing mutagenesis. To date, several hundred mutations have been made inL. casei, E. coli and human TS (Climie et al., 1990b; Michael et al.,1990). Most of mutations in L. casei were produced by cassettemutagenesis (Wells et al., 1985; Climie et al., 1990a). The synthetic L.casei TS gene was engineered by creating over 30 unique restrictionsites about equally spaced throughout the entire gene, providing“replacements sets” in which several target amino acids were replaced bya large number of substitutions. Another approach involving theintroduction of an amber stop codon were adopted to generate multiplemutants of E. coli TS (Michaels et al., 1990; Kim et al., 1992). Usingthese approaches the various mutants in either L. casei or E. colisystem were first screened for catalytic activity of TS by geneticcomplementation in a TS-deficient E. coli host, and then mutants ofinterest characterized by kinetic studies. A few mutants of human TS andtheir expressed enzymes in mammalian cells have also been studied. Themutant human TSs were also tested to complementation of the growth ofTS-negative E. coli stains in the absence of thymine to determine if theactivity of an altered enzyme is sufficient to support growth. However,the correlation of a mutant human TS and drug resistance can not beinterpreted by this complementation study in a bacterial system andmammalian cells lacking TS are required. The three-dimensional structureof human TS has provided the impetus to generate mutants of human TShaving novel enzyme properties such as drug resistance.

Prediction of properties of enzymes obtained by site-directed mutationsis poor. When the enzyme accommodates a single amino acid substitution,readjustment of neighboring residues may occur, resulting in structuralplasticity. TS is one of best examples for observing this phenomenon. Ingeneral, TS can tolerate amino acid substitutions even in a highlyconserved residue that is important for enzyme structure or function. Ina few cases, a single amino acid replacement causing dramatic change inproperties of TS was also found. By reviewing the mutations alreadymade, it was found that highly conserved residues are hot spots foramino acid substitutions (Carreras et al., 1995), and there are a fewresidues such as Arg50, Glu87, Trp109, Cys195, Arg215, Asp218, andTyr258 especially sensitive to substitution (Stroud et al., 1993). Allof these residues are in the substrate or folate binding site.

Cys195 (ec146, lc198) involved in the binding of 2′-deoxyuridylate aswell as initiating the catalytic process could only be modified to Serfor E. coli TS and still retain activity, albeit severely diminishedactivity. None of the comparable L. casei mutants showed detectableactivity (Dev et al., 1988; Climie et al., 1990b). Conserved Argresidues at positions 50, 215, 175, and 176 form a positively chargedbinding surface for the phosphate anion of dUMP. In L. casei, ForArg175, another completely conserved residue could be replaced by aneutral (Ala, Thr), positive (Lys). or negative (Glu) amino acid withoutdrastic changes in substrate binding or catalytic activity (Santi et al,1990). Most substitutions for Arg176 of either E. coli or L. casei TSresult in little impairment of function. In contrast, Arg218 could notresist any amino acid shifts.

The Arg50 loop, having less than 1.0 Å movement and reorientation uponArg50 (ec21, lc23) binding to the phosphate of dUMP, is a highlyconserved region. For Arg50, only four amino acids (Gly, Pro, Ser, andHis) in E. coli TS and three residues (Val, Ile, and Gln) in L. casei TSare substitutable with retention of 10-50% of the wild-type activity(Zhang et al., 1990; Michaels et al., 1990). Asp49 (ec20, lc22) is quitesensitive to mutagenesis, except for replacements by the two polar (Cys,Ser) and one acidic (Glu) residues, all E. coli Asp49 mutants do notcomplement growth of TS-negative cells. In E. coli TS, Thr51 (ec22,lc24) tolerates substitutions of Pro, Ser, Tyr, Gln and Lys.Surprisingly, contrary to those neighbor residues, Gly52 (ec23, lc:His52) accepts any mutations. This residue has apparent reorientationupon the formation of ternary TS complex (Kim et al., 1992).

Trp109 (ec80, lc82) and Asn112 (ec: Trp83, lc: Trp85) are highlyconserved residues that form hydrophobic contacts with both dUMP andCH₂H₄folate. Trp109 activity could not be fully restored by any of thesubstitutions except phenylalanine for E. coli TS, but showed highactivity by three amino acid (Phe, Tyr and His) changes for L. casei TS(Michaels et al., 1990). Asn112 was only mutated to Phe for L. casei TSand the altered enzyme remained functional, but the W109F/N112F doublemutant of L. casei was inactive (Carreras et al., 1995). Phe59 (ec30,lc32) forms part of the substrate binding pocket in tertiary structure.Leu and Tyr replacements for Phe59 of E. coli TS yield enzymes thatcomplement the TS-deficient E. coli strain, but TS activity was totallylost for other substitutions (Kim et al., 1992).

The C-terminus region of TS plays a critical role in folate binding andcatalysis (Perry et al., 1993). Deletion of just the residue Val313results in TS protein that can bind both ligands but is catalyticallyinactive because the protein is incapable of closure to sequester thereactants. However, Val313 could tolerate almost all substitutions andmany mutants were as active as wild-type TS (Climie et al., 1992;Carreras et al., 1992). A few mutagenesis studies for human TS have beenpublished. Gln214, being believed in a kink region for three (β-sheetformation of the central core of the polypeptide, is highly conserved inall TSs. Cell growth of the TS-negative E. coli was supported by Glu,His, Lys, or Ala, but not by Ser, Cys, or Trp substitutions (Zhao etal., 1995).

Until recently, only one mutation in TS has been reported to be relatedto TS-directed drug resistance. Tyr33 of human TS is one of 40 aminoacid residues that are invariant among all reported TS sequences. TheTyr33 to His33 substitution was discovered in a human colon tumor cellline and conferred approximately a 3- to 4-fold resistance to FdUMP, ametabolite of the chemotherapeutic prodrug 5-fluorouracil. This mutationaffects the catalytic properties of the TS enzyme, showing an 8-folddrop in k_(cat) for the reaction. The K_(m) values for both dUMP andCH₂H₄folate were not significantly different between the mutant andwild-type TS.

The crystal structure of human TS has shown that the side chain of Tyris not directly involved in ligand binding site of the human TS.However, the hydroxyl oxygen of Tyr33 is hydrogen bonded to the backbonecarbonyl oxygen of residue 219 at the first turn of the centralhydrophobic helix J (residues 219-242). The first turn of helix J isconsisted of eight amino acid residues (219-226), five of which arehighly conserved and two (Leu221 and Phe225) form a hydrophobic pocketfor the PABA ring of the cofactor. The drug-resistant mutation can beinterpreted in terms of induced change by reorientation in the initialturn of the helix J to be no longer optimal for ligand binding. Why somesubstitutions are active in E. coli but not in L. casei TS, and viceversa is not known.

The discovery and development of TS inhibitors was based on molecularstructures and properties of TS and its pyrimidine substrate or folatecofactor, especially as the three-dimensional structures of severalunliganded and liganded TSs at the atom level of resolution wereachieved. The first compounds to have clinically significantTS-inhibiting activity were the fluoropyrimidines 5-FU and FdUrd, whichare metabolized to 5-fluorodeoxyuridine monophosphate (FdUMP) thatsubsequently occupies the substrate binding site leading to a stable andinactive TS complex. In addition, they also may be incorporated into RNAor DNA via fluoro-UTP or 5-fluoro-dUTP, respectively. Therefore,fluoropyrimidines are not pure TS inhibitors and are susceptible tometabolic degradation in vivo. In contrast, folate analogues may bedesigned as more specific and more stable TS-specific inhibitors.Moreover, the cofactor CH₂H₄folate is a relatively large molecule, whichprovides a variety of sites, amenable to manipulation in drug design(Schoichet et al., 1993).

ZD1694 and BW1843U89 are new, promising antifolates that are derivedfrom the CB3717 chemical scaffold, which are characterized as classicalantifolate TS inhibitors. They contain a glutamate moiety and can bemetabolized to noneffluxable polyglutamate forms within the cell. Thepolyglutamylated TS inhibitors bind tighter than the correspondingmonoglutamylated forms. By comparison, the nonclassical antifolate TSinhibitors. such as AG337 and AG331, lacking the glutamate, haverecently been developed.

The antineoplastic agent 5-FU is a mechanism-based inhibitor of TS,which is metabolized to FdUMP that forms a stable covalent adduct withCH₂H₄folate as a steady-state intermediate, resulting in inhibition ofTS. CB3717 (N¹⁰-propargyl-5, 8-dideazafolic acid), a lead compound asanalogue of CH₂H₄folate, is a 2-amino-4-hydroxy quinazoline carrying apropargyl group on N-10 that greatly increases the affinity of TS, withK_(i) of 2.7 nM. CB3717 demonstrated antineoplastic activity in Phase Itrials, but its development was abandoned due to unpredictable severerenal and hepatic toxicity caused by its poor aqueous solubility.Tomudex(N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino-]-2-thenoyl)-L-glutamic acid) was developed based on the molecularstructure of CB3717. This quinazoline folate analogue, designed to bemore water-soluble than CB3717 to avoid some side-effects such asnephrotoxicity, is a highly selective inhibitor of mammalian TS (Jodrellet al., 1991). Similar to CB3717, tomudex results in decreased TMPproduction, which leads to inhibition of DNA synthesis, resulting incell death (Jackman et al., 1991a, b, & c).

Tomudex is a mixed noncompetitive TS inhibitor. In contrast to CB3717,tomudex enters cells using the reduced folate carrier (RFC). In the cellit is an excellent substrate for FPGS with an affinity 30 times higherthan that of CB3717, and it is rapidly polyglutamylated by FPGS. Thepolyglutamated forms (n=2-6) are up to 100-fold more potent inhibitorsof TS than is the monoglutamate. The polyglutamates are retained withincells, leading to a prolonged inhibitory action even in the absence ofextracellular compound (Jackman et al., 1993). Tomudex thus is 500-foldmore active in inhibiting cell growth than CB3717, despite being 20times less potent as a TS inhibitor in enzyme assays (K_(i), 60 nM)(Gibson et al., 1993; Lu et al., 1995). Tomudex has demonstratedactivity in colorectal, breast, and pancreatic cancer and was approvedin the U.K. for treatment of advanced colorectal cancer in August 1995.Also, phase III trials of tomudex in advanced colorectal cancer showedthat tomudex is slightly superior to 5-FU with respect to anti-advancedcolorectal cancer activity and therapeutic margin. BW1843U89 is anextremely potent, noncompetitive TS inhibitor in enzyme assays (Ki, 90pM). As a TS-directed inhibitor, the monoglutamated form of BW1843U89 isas potent as the polyglutamated derivatives of tomudex in vitro studies.Similar to tomudex, growth inhibition could be reversed by thymidinealone, indicating that TS is its exclusive site of action. BW1843U89does not require the RFC for the cellular entrance and is an excellentsubstrate for FPGS, but is only metabolized to a diglutamated form. Thepolyglutamation of this antifolate leads to retention in cells.

Drug resistance is a major obstacle to the successful use ofchemotherapeutic agents in the treatment of neoplastic disease. Formaintaining efficacious drug therapy, discovering new antitumor agentsis an important goal. For drug resistance, investigations of naturallyoccurring resistance in model cell lines provides insights into themechanisms that underlie innate clinical resistance in patients notpreviously exposed to these new drugs. The prior art is deficient in thelack of effective means of inhibiting the overcoming the resistance toTS inhibitors routinely encountered in anti-neoplastic therapy. Thepresent invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

The present invention randomly mutated HT1080 cells and subsequentlyselected drug-resistant clones with a high concentration of AG337.Secondly, site-directed mutagenesis was performed on three codons thatcode for amino acids that are folate-binding sites of human TS gene,based on the knowledge of three-dimension structures of TS. Using thesetwo approaches, isolation and characterization of mutants of human TSconferring drug resistance to TS specific inhibitors were studied. Thehuman TS mutants obtained have desirable properties including antifolateresistance, a high catalytic efficiency and good stability. This kind ofTS variant is an excellent candidate for gene therapy approaches, namelyto transfer drug resistance to human hemotopoietic progenitors, thusallowing dose-intense therapy in cancer patients by protecting normalcells and preventing dose-limiting myelotoxicity. Moreover, thesemutants may be used as dominant selectable markers in therapeutic genetransfer protocol.

In one embodiment of the present invention, there is provided a mutatedhuman thymidylate synthase, said mutated synthase differing from wildtype thymidylate synthase of the amino acid sequence disclosed inGenbank Accession number NP001062 (SEQ ID No. 39) at amino acid residue49, amino acid residue 52, amino acid residue 108, amino acid residue221 or amino acid residue 225.

In another embodiment of the present invention, there is provided a cDNAencoding the mutated human TS of the present invention.

In yet another embodiment of the present invention, there is provided aDNA vector comprising: DNA encoding a mutated human TS of the presentinvention.

In still yet another embodiment of the present invention, there isprovided a host cell transfected with the DNA vector of the presentinvention and wherein said host cell produces a mutated human TS.

In still yet another embodiment of the present invention, there isprovided a method of decreasing the toxic effects of anti-neoplasticinhibitors of TS in an individual in need of such treatment, comprisingthe steps of: introducing a mutated human TS into cells of saidindividual; and returning said cells to said individual.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows the principle of single-strand conformation polymorphism(SSCP).

FIG. 2 shows an autoradiogram of SSCP analysis of human TS genemutations in AG337-resistant cells. Amplified DNA fragments by RT-PCRcorresponding to region A were denatured by heating and electrophoresiswas performed in 8% polyacrylamide gel containing 5% glycerol atconstant 30 W at 4° C. Lane 1: control HT1080 cells; Lane 2 to Lane 5:AG337-resistant cells. Fragments with mobility shift in addition towild-type bands are observed in Lane 4 and Lane 5, suggesting mutationsin human TS gene from nucleotide coding 70 to 187 for HT1080/1b andHT1080/2b.

FIG. 3 shows the northern blot analysis of total RNA isolated from 10resistant sublines and parental HT1080 cell line. Upper panel: total RNA(20 micrograms) was electrophoreses, blotted onto a nylon membrane, andhybridized with the [³²P]dCTP-labeled human TS cDNA. Lane 1: RNA fromparental HT1080 cells. Lane 2: RNA from resistant cell line HT1080-Awhich was selected by AG337 without EMS pretreatment. Lanes 3-11:represent 9 different resistant sublines from EMS treatment and followedby AG337 selection. Lower panel: membrane was stripped and rehybridizedwith a [³²P]dCTP-labeled 36B4 ribosomal control cDNA probe.

FIG. 4 shows the western blot analysis of human TS protein levels inparental HT1080 cell lines and 10 AG337-resistant cell lines. Westernblot analysis for human TS was carried out using 100 micrograms ofcellular lysate fractionated on 12% acrylamide gel. A rabbit antibodyagainst human TS was used as a probe.

FIG. 5 shows the sequencing analysis of parental cell line HT1080 andresistant cell line HT1080/1e for human TS cDNA. The region detectedbetween nucleotide coding 147 and 162 is shown. A point mutation occusat 154 (G→A), resulting in Ser⁵² instead of Gly⁵².

FIG. 6 shows the graphical comparison of the IC50 values for human TSmutants for tomudex (ZD1694). IC50 values were determined by Alamar Blueassay. Error bars denote the standard deviation of multiple IC50determinations.

FIG. 7 shows the graphical comparison of the IC50 values for human TSmutants for AG337. IC50 values were determined by Alamar Blue assay.Error bars denote the standard deviation of multiple IC50determinations.

FIG. 8 shows the graphical comparison of the IC50 values for human TSmutants for BW1843U89. IC50 values were determined by Alamar Blue assay.Error bars denote the standard deviation of multiple IC50determinations.

FIG. 9 shows the graphical comparison of the IC50 values for human TSmutants for FdUrd. IC50 values were determined by Alamar Blue assay.Error bars denote the standard deviation of multiple IC50determinations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a mutated human thymidylatesynthase, said mutated synthase differing from wild type thymidylatesynthase of the amino acid sequence disclosed in Genbank Accessionnumber NP001062 (SEQ ID No. 39) at amino acid residue 49, amino acidresidue 52, amino acid residue 108, amino acid residue 221 or amino acidresidue 225. In one mutated form, the amino acid residue 49 is mutatedto an amino acid selected from the group consisting of asparagine andglycine. In another mutated form, amino acid residue 52 is mutated toserine. In another mutated form, amino acid residue 108 is mutated to anamino acid selected from the group consisting of alanine, phenylalanine,glycine, glutamic acid and asparagine. In another mutated form, aminoacid residue 221 is mutated to an amino acid selected from the groupconsisting of phenylalanine, arginine, alanine, isoleucine and serine.In another mutated form, amino acid residue 225 is mutated to an aminoacid selected from the group consisting of tryptophan, serine, leucineand tyrosine.

The present invention is also directed to a cDNA, said cDNA encoding amutated human TS. The present invention is also directed to a DNA vectorcomprising: DNA encoding a mutated human TS. The present invention isalso directed to a host cell transfected with the DNA vector wherein thehost cell produces a mutated human TS. Preferably, the host cell is amammalian hematopoietic cell and most preferably, the host cell is aperipheral blood stem cell.

The present invention is also directed to a method of decreasing thetoxic effects of anti-neoplastic inhibitors of TS in an individual inneed of such treatment, comprising the steps of: introducing a mutatedhuman TS into cells of said individual; and returning said cells to saidindividual. Preferably, but not exclusively, the inhibitor of TS isselected from the group consisting of 5-fluorouracil, N¹⁰-propargyl-5,8-dideazafolic acid,N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino-]-2-thenoyl)-L-glutamic acid, ZD1694 and BW1843U89.

The mutated human TSs may also be used as a selectable marker. Forexample, the present invention also provides a method of selecting amongclones for the introduction of a non-selectable gene, comprising thesteps of: (a) inserting the non-selectable gene into a DNA vectorcomprising DNA encoding a mutated human TS; (b) introducing the vectorinto cells of a type in which the non-selectable gene and the mutatedhuman TS are expressed; and (c) selecting cells which are resistant toinhibition by anti-neoplastic inhibitors of TS.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1 Materials

The pCR-Script™ SK(+) cloning kit was purchased from Stratagene (LaJolla, Calif.). Transformer™ site-directed mutagenesis kit was purchasedfrom CLONTECH Laboratories, Inc. (Palo Alto, Calif.). DOTAP was suppliedby either Boehringer Mannheim (Indianapolis, Ind.) or by the liposomefacility at Cornell Medical College (Dr. T. Scotto, New York, N.Y.). TheGeneClean kit was obtained from BIO 101 Inc. (La Jolla, Calif.). DNASequencing kit and IPTG were purchased from United States BiochemicalCorp. (Cleveland, Ohio). The bacterial expression plasmid pET-17(b andcompetent E. coli BL21(DE3) cells were from Novagen, Inc. (Madison,Wis.). The mammalian expression vector pcDNA3 was from Invitrogen, Corp.(San Diego, Calif.). DEAE-cellulose (DE52) was from Whatman (Clifton,N.J.), and Phenyl Sepharose CL-4B was from Pharmacia Biotech(Piscataway, N.J.). Bovine serum albumin (BSA) and Ethylmethanesulfonate(EMS) were supplied by Sigma Chemical Co. (St. Louis, Mo.). Fetal bovineserum and molecular weight standards were purchased from Gibco BRL(Gaithersburg, Md.). Bacterial growth and tissue culture media weresupplied by an in house media unit. Restriction enzymes and DNAmodifying enzymes were purchased from various suppliers (New EnglandBiolabs, Bio-Rad, Promega, Stratagene, and Pharmacia). Ampli-Taqpolymerase was purchased from Perkin-Elmer (Norwalk, Conn.). Chemicalswere obtained from commercial sources and used without purification.

EXAMPLE 2 DNA Oligonucleotides

Oligonucleotide primers were synthesized by either IDT, Inc.(Coralville, Iowa) or Operon Technologies, Inc. (Alameda, Calif.). Priorto synthesis, all oligonucleotide sequences requested were checked withthe computer program Oligo (Version 4.0.2., National Biosciences, Inc.)to optimize the primer design.

EXAMPLE 3 Source of Human TS Plasmid

Human recombinant TS cDNA of the sequence disclosed in Genbank Accessionnumber NM001071 (SEQ ID No. 38) in bacterial expression vectorpET-17(b), named pET-17(bhTS), was provided by Dr. Frank Maley. In orderto increase the levels of human TS protein expression in E. coli, thecodon usage in the 5′-coding region of the gene was modified in thisconstruct. The sequence ATGCCTGTGGCCGGC (SEQ ID NO:1) was changed toATGCTTGTTGCIGGT (SEQ ID NO:2). These changes resulted in an alterationof Pro2 to Leu2. However, both in vivo and in vitro experiments provedthat this substitution does not result in functional change for human TSenzyme.

EXAMPLE 4 Substrates and Inhibitors of Human TS Enzyme

AG337 was a gift of Agouron Pharmaceuticals, Inc. (San Diego, Calif.).CH₂H₄folate was synthesized by Schircks Laboratories (Switzerland).Tomudex was obtained from Zeneca (Macclesfield, United Kingdom).BW1843U89 was supplied by Glaxo-Wellcome (Research Triangle park, N.C.).dUMP, FdUrd, and FdUMP were purchased from Fisher Scientific (Fairlawn,N.J.). The concentrations of compounds were determined by UV absorbance,using appropriate extinction coefficients.

EXAMPLE 5 Cell Lines and Culture Conditions

Human fibrosarcoma HT1080 cells were obtained from the American TypeCulture Collection (Rockville, Md.). Stock cultures of the parental cellline HT1080 and resistant sublines were maintained in RPMI 1640 mediumsupplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 μg/mlstretomycin, and 100 units/ml penicillin.

The TS-negative cell line FSthy21, a gift of Dr. T. Seno (Ayusawa etal., 1981), was originally established from mouse FM3 A cells. FSthy21cells were grown in Eagle's minimum essential medium supplemented with10% dialyzed fetal bovine serum, 2 mM L-glutamine, 100 μg/mlstretomycin, 100 units/ml penicillin, 1 μM reduced folate and 10 μMthymidine. Prototrophic transformant clones, derived from FSthy21 cellsby transfection of wild-type or mutant human TS cDNAs, were cultured inthe same medium as mentioned above without thymidine and reduced folatesupplements.

EXAMPLE 6 Chemical Mutagenesis and Drug Selection: Ethylmethanesulfonate(EMS) Sensitivity of HT1080 Cells

HT1080 cells were seeded into 6-well plates (100-300 cells/well). Afterovernight incubation to allow cell attachment, The cells were exposedwith EMS for 18 hours, washed three times, and then incubated withEMS-free medium for a further 14 days. The range of EMS concentrationtested was 100 to 1000 μg of EMS/ml of culture medium. Colonies formed(50 cells) were stained with crystal violet and counted. Theconcentration of EMS used for the random mutagenesis experiments wasdetermined based on the number of colonies obtained in the EMS-treatedtest versus EMS concentrations (Sega, 1984; Fanin et al., 1993).

EXAMPLE 7 AG337 Sensitivity of HT1080

The optimum concentration of AG337 for selection of resistant clones wasdetermined as follows. The human fibrosarcoma HT1080 cells were seededinto 6-well plates at 100 cells per well and incubated at 37° C. withcomplete medium. Various concentrations of AG337 were added after 24hours. Drug-containing medium was changed every 4 days and survivingcolonies were counted after 14 days of drug treatment. IC₅₀ values andthe minimal concentration of AG337 that resulted in no colony formationin HT1080 cells were obtained by plots of colonies surviving versusAG337 concentrations.

EXAMPLE 8 Chemical Mutagenesis and Drug Selection

HT1080 cells (4×10⁸) growing in logarithmic phase were exposed 18 hr toEMS (400 μg/ml, a concentration that resulted in 80% inhibition ofcolony formation). The cells were washed and incubated a further 3 daysin EMS-free medium to allow phenotypic expression. EMS-treated cellswere subcultured at 6×10⁷ cells/100 mm dishes, and then grown in thepresence of 40 μM AG337 for 14 days. Forty-one individual clonesobtained from EMS and AG337 sequentially treated cells were isolatedwith a ring cylinder and expanded into stable resistant sublines.Without EMS pretreatment, only 1 clone (1×10⁸ cells were plated as acontrol) survived exposure to 40 μM AG337.

EXAMPLE 9 Isolation of Total RNA

For Northern blot analysis and RT-PCR, the cells from HT1080 and 41resistant sublines in log phase were harvested from 150-cm² tissueculture flasks, and RNA isolated using RNAzol (Bioteox lab., Inc.)according to the manufacturer's instructions. RNA pellets wereresuspended in RNAse and DNAse free water and quantitatedspectrophotometrically (OD₂₆₀=33 μg RNA/ml). The ratio of OD₂₆₀/OD₂₈₀was greater than 1.8 in all of samples.

EXAMPLE 10 Reverse Transcription for cDNA Synthesis

Total RNA samples were heated in water for 5 minutes at 65° C. and thenquickly cooled in an ice-water bath. For each sample, 5 μg of total RNAwas adjusted to a final volume of 20 μl. The reaction mixture wascomposed of 50 mM Tris-HCl, pH 8.3; 50 mM potassium chloride; 6 mMmagnesium chloride; 10 mM DTT; 10 μg random primer pd(N)₆; 50 units/mlRNAsin; 500 μM dNTPs; and 200 units AMV reverse transcriptase. Thesynthesis of first strand cDNA was performed at room temperature for 5min. and then switched to 42° C. for 1 h after which the samples wereused or stored at 20° C.

EXAMPLE 11 PCR Amplification, DNA-SSCP Screening and Sequence Analysisfor Detection of Mutations: Polymerase Chain Reaction

The PCR amplification was performed in DNA thermal cycler for 40 cyclesin a 50 μl of total volume containing 5 μl of the reaction mixture ofreverse transcription as the template, 50 pmols of each primer (primersequences used for PCR amplification were sense5-CACAGGAGCGGGACGCCGAG-3′ (SEQ ID NO:3) and antisense5′-AACAGCCATTTCCATTTTAATAGT-3′ (SEQ ID NO:8), which cover a 890 bp offragment from nt 50 to the C-terminus of the human TS gene), 80 μM ofeach of the four dNTPs, 1×PCR buffer and 1 unit of Taq DNA polymerase.After 1 cycle of initial denaturing at 94° C. for 10 min., annealing at55° C. for 1 min., and extension at 72° C. for 2 min., 40 cycles of 94°C. for 1 min., 55° C. for 1 min., and 72° C. for 2 min were performed.The PCR product corresponding to a 890 bp fragment was separated on a1.2% TAE agarose gel and identified with ethidium bromide. The amplifiedDNA fragments were purified using a Gene Clean kit. The sequences ofoligonucleotide primers which were used in various experiments such asPCR amplification, DNA-SSCP and sequence analysis are described in TABLE1.

TABLE 1 The Sequences of Oligonucleotides Used for PCR Amplification.DNA-SSCP Screen and Sequence Analysis Oligonucleotide Sequence ofOligonucleotide Anneals to Reference 5′ → 3′ human TS hTS-1A*CACAGGAGCGGGACGCCGAG nt 50 to 69 (SEQ ID NO: 3) hTS-1BCAAAAGTCTCGGGATCCATT nt 354 to 334 (SEQ ID NO: 4) hTS-2AGAGCTGTCTTCCAAGGGAGTGA nt 298 to 319 (SEQ ID NO: 5) hTS-2BTCTCTGGTACAGCTGGCAGGACAG nt 645 to 622 (SEQ ID NO: 6) hTS-3ACTGCCAGTTCTATGTGGTGAACAGTG nt 594 to 616 (SEQ ID NO: 7) hTS-3BAACAGCCATTTCCATTTTAATAGT nt 939 to 915 (SEQ ID NO: 8) hTS-4ATACCTGGGGCAGATCCAACAC nt 97 to 117 (SEQ ID NO: 9) hTS-4BTTCATCTCTCAGGCTGTAGCGCG nt 210 to 188 (SEQ ID NO: 10) hTS-5ATCAGATTATTCAGGACAGGGAGTTG nt 451 to 475 (SEQ ID NO: 11) hTS-5BATGGTGTCAATCACTCTTGCAG nt 503 to 481 (SEQ ID NO: 12) hTS-6AGGGAGATGCACATATTTACCTGAA nt 756 to 779 (SEQ ID NO: 13) hTS-6BTCTGGGTTCTCGCTGAAGCT nt 822 to 803 (SEQ ID NO: 14) *Capital A representssense, and B represents antisense

EXAMPLE 12 Single-Stranded Conformation Polymorphism (SSCP) Analysis

For DNA-SSCP analysis, small human TS fragments (150-260 bp) wereobtained by PCR amplification of TS cDNA using 6 pairs of TS specificprimers (see TABLE 2). The reaction mixture containing 1 μCi [α-³²P]dCTPand a small volume (3 μl) of final PCR products was subsequently mixedwith 10 μl of loading buffer containing 96% formamide. Samples weredenatured at 94° C. for 3 min., chilled on ice for at least 5 min. and 2μl was loaded onto a 6-8% non-denaturing polyacrylamide gel with orwithout 10% glycerol. Gels were electrophoresed at 10 W for 6-8 hours at4° C., using 0.5 (Tris-borate-EDTA buffer. The separated single-strandDNA fragments were visualized by autoradiography.

An alternative method (called nonisotopic SSCP) was also utilized, inwhich the single-strand DNA bands are detected by ethidium bromidestaining instead of autoradiography. At least 40 ng (20 μl) of amplifiedDNA fragments was denatured by addition of 1 μg 0.5 M NaOH, 10 mM EDTAat 42° C. for 5 minutes. Just before loading, 1 μl of formamidecontaining 0.5% bromophenol blue and 0.5% xylene cyanol were added.Non-denaturing gels (1.5 mm thick, 6-8% polyacrylamide), with andwithout 5% glycerol, were made in a standard vertical gel apparatus.Gels, using 0.5(TBE as running buffer, were electrophoresed at 15 (V/cmof gel) for 4 hours and temperature was maintained at 4° C. bycirculating cold water. Finally, SSCP gels were neutralized and stainedin 0.5×TBE containing 0.5 μg/ml ethidium bromide for band observation.The principles of SSCP for detection of mutations is presented by FIG.1.

EXAMPLE 13 pCR-Script™ SK(+) Cloning

The RT-PCR fragments with putative mutations in human TS weresubsequently subcloned into pCR-Script™ SK(+) vector, which permits theefficient cloning of PCR fragments with a high yield and a low rate offalse positives. After ligation and transformation, the positive whitecolonies containing human TS fragments were chosen for further miniprepisolation and sequence analysis.

EXAMPLE 14 DNA Sequence Analysis

Sequence analysis was performed by the dideoxy chain termination methodwith α-³⁵S dATP using modified T7 DNA polymerase according to themanufacture's instructions. Templates were either PCR products oralkali-denatured plasmid DNA. The plasmid ligated by pCR-Script™ SK(+)Cloning vector with human TS fragments exhibiting abnormal mobility onSSCP gels were sequenced by two commercial designed primers [M3 (−20)and M13 reverse]. Direct sequencing of the PCR products was carried outusing single-stranded DNA products denatured by NaOH as templates anddesigned human TS oligonucleotides (see TABLE 1) as sequencing primers.After completion of the sequencing reaction, the products were loaded ona 8% polyacrymide urea gel, which was electrophoresed at 80 W for 2-3hours after which the gel was dried and exposed to film. Mutations wereverified by comparison of wild-type control lanes

EXAMPLE 15 Studies of AG337-Resitant Cell Lines: Whole Cell TS Assay

Resistance to TS inhibitors was evaluated using the whole cell TS assayfor several AG337-resistant sublines and parental cell line HT1080 as acontrol. Cell suspensions (2×10⁶ cells/ml) were exposed for 4 hours todifferent concentrations of either an antifolate drug (AG337, tomudex)or 5-fluoro-2′-deoxyuridine (FdUrd). For the absence of TS inhibitors,cells convert 2′-[5-³H]-deoxyuridine to dTMP, releasing ³H₂O into themedium. The rate of dTMP synthesis in the presence or absence of drugwas assessed at 0, 15, 30, 40 minutes, by measurement of [3H]₂O releaseby charcoal-TCA (trichloroacetic acid) separation of ³H₂O fromnucleosides and nucleotides. Radioactivity was quantitated using aliquid scintillation counter (Beckman LS5).

EXAMPLE 16 Northern Blot Analysis

Fifty μg samples of RNA were subjected to electrophoresis through 1.5%denaturing formadehyde-agarose gels in 1×MOPS buffer, running atconstant volts (5 V/cm of gel). The gels were washed in water and 10×SSCand then transferred onto a nitrocellulose membrane. After blotting, RNAon the membrane was immobilized by UV cross-linking, prehybridized for24 hours at 42° C., and then hybridized at the same temperature for afurther 18 hours. The human TS probe, which was ³²P-labeled by randomprimer DNA labeling kit, was a 950 bp gel-purified fragment of human TScDNA cleaved from the pET-17×bhTS plasmid with NdeI and HindIIIrestriction enzymes. Ribosomal phosphoprotein 36B4 cDNA was used as aloading control. After hybridization, the blots were washed andvisualized by autoradiography.

EXAMPLE 17 Western Blot Analysis

Protein concentrations in cell extracts were determined by the Bradfordassay using Bio-Rad dye reagent. Protein extracts (100 μg) were boiledfor 5 minutes, loaded on a 12.5% polyacrylamide-SDS gel, and thenelectrophoresed at constant current (30 mA/gel). The gel was transferredonto a nitrocellulose membrane, which was then blocked overnight with 5%milk in 0.1 M Tris, pH 7.4. To detect TS, a rabbit anti-human TSpolyclonal antibody at a 1:1000 dilution in the same buffer as above wasadded and the blots were washed with 0.1 M Tris, pH 7.4. The goatanti-rabbit IgG antibody at a dilution of 1:500 was used as thesecondary antibody. Human TS proteins were visualized by the ECL method.The polyclonal antibody for human TS was a gift of Dr. Frank Maley.

An alternative more rapid hydrophobic blotting procedure was also usedby utilizing immobilon-P membrane instead of the nitrocellulosemembrane. After transfer, the membrane was soaked in 100% methanol for 2min. and then allowed to air dry for 15 minutes. The completely driedmembrane was incubated with the TS antibody in the buffer (1×saline, 1%non-fat milk and 0.05% Tween-20) at 4° C. for 1 hr with gently shaking,washed with buffer (lxsaline and 0.05% Tween-20) at room temperature for2 min. twice, and incubated with goat anti-rabbit IgG antibody in thesame buffer and incubated, following washing, the protein bands werevisualized.

EXAMPLE 18 Construction of a Mammalian Expression Vector for Human TS

The vector pcDNA3 under T7 promoter control was used to construct amammalian expression system for human TS. The construction of theplasmid containing recombinant human TS cDNA of the sequence disclosedin Genbank Accession number NM001071(SEQ ID No. 38) was performed bydigestion of pET-17xbhTS with NdeI and HindIII to generate a 950 bpfragment containing a full cDNA sequence of human TS. The reactionmixture was treated with T4 DNA polymerase to make blunt ends andisolated by electrophoresis on a 1.2% TAE agarose gel. This 950 bp DNAfragment was cut out and then purified by use of the GeneClean kit. ThepcDNA3 expression vector was digested with EcoRV and the sole DNAfragment (5.4 kb) was extracted. The pcDNA3 fragment was then mixed witha 10-fold excess of the 950 bp fragment in the presence of T4 ligase andincubated at 14° C. overnight. The ligation mixture was then directlytransformed into competent DH5αcells. A pcDNA3hTS plasmid with correctsize (6.4 kb) and orientation was confirmed by restriction mapping andsequencing.

EXAMPLE 19 Site-Directed Mutagenesis

The Transformer™ site-directed mutagenesis kit was used to obtain pointmutations in human TS (2nd version, Clontech). This single-strand basedtechnique takes advantage of the difference in the transformationefficiency between circular and linear DNA. Twenty-two oligonucleotideswere designed and synthesized. The last one (TABLE 2) is a selectionprimer that contains a unique KspI restriction site instead of theunique SmaI restriction site on the pcDNA3 vector. The other 22 primerswere designed to obtain mutants of the human TS gene at the targetedsite. Mutant human TS cDNA was obtained by annealing one mutagenic andone selection primers to the alkali-denatured single-strand pcDNA3hTSplasmid. The second strand DNA was synthesized by T4 DNA polymerase andcycled by T4 DNA ligase using the pcDNA3hTS as a template and the twoannealed primers. The reaction mixture, which contained circular DNAwith one mutated and one unchanged strand, was digested with SmaI andthen transformed into DNA repair-deficient BMH 71-18 mutS cells.Transformed bacteria cells were grown overnight and the mixed plasmidswere isolated by miniprep. In order to increase the transformationefficiency of the circular plasmid containing the mutant human TS, theplasmid mixture was linearized with SmaI again and subsequentlytransformed into E. coli DH5(competent cells and plated. After doubleSmaI digestion and double transformations, the resulting colonies werescreened using KspI digestion that only cuts newly synthesized plasmids.Plasmids with putative point mutations in human TS were examined furtherby restriction mapping and DNA sequencing.

TABLE 2 Oligonucleotides Used for Site-Directed Mutagenesis (NucleotideUnderlined were those used to change the codon) Position in hTSMutagenic oligonucleotide Reference and created sequences number &mutation 5′ → 3′ SEQ ID NO. Phe²²⁵→Trp²²⁵ CGGTGTGCCTT GG AACATCGCCAG162 (SEQ ID NO: 15) Phe²²⁵→Ser²²⁵ CGGTGTG C CTCCCAACATCGCCAG 163 (SEQ IDNO: 16) Phe²²⁵→Leu²²⁵ CTCGGTGTGCCT C TCAACATCGCC  83 (SEQ ID NO: 17)Phe²²⁵→Tyr²²⁵ CGGTGTGCCT TA CAACATCGCCAG  84 (SEQ ID NO: 18)Leu²²¹→Phe²²¹ GGAGACATGGGC T TCGGTGTGCCTT 164 (SEQ ID NO: 19)Leu²²¹→Arg²²¹ GAGACATGGGCC G CGGTGTGCCTTTC 165 (SEQ ID NQ: 20)Leu²²¹→Ala²²¹ GAGACATGGGC GC GGTGTGCCTT  85 (SEQ ID NO: 21)Leu²²¹→Ile²²¹ GAGACATGGGC A TCGGTGTGCC  86 (SEQ ID NO: 22) Leu²²¹→Ser²²¹GAGACATGGGC AG CGGTGTGCCTT  87 (SEQ ID NO: 23) Ile¹⁰⁸→Ala¹⁰⁸ GGGAGTGAAAGC CTGGGATGCC 175 (SEQ ID NO: 24) Ile¹⁰⁸→Phe¹⁰⁸ CAAGGGAGTGAAA TTCTGGGATGCCA 176 (SEQ ID NO: 25) Ile¹⁰⁸→Gly¹⁰⁸ GGGAGTGAAA GG CTGGGATGCC270 (SEQ ID NO: 26) Ile¹⁰⁸→Gly¹⁰⁸ GGGAGTGAAA GAG TGGGATGCC 271 (SEQ IDNO: 27) Ile¹⁰⁸→Asn¹⁰⁸ GGGAGTGAAA AA CTGGGATGCC 272 (SEQ ID NO: 28)Asp⁴⁹→Asn⁴⁹ GTCAGGAAGGAC A ACCGCACGGGCA 923 (SEQ ID NO: 29) Asp⁴⁹→Gly⁴⁹TCAGGAAGGACG G CCGCACGGGCAC 928 (SEQ ID NQ: 30) Thr⁵¹→Ala⁵¹ AAGGACGACCGCG CGGGCACCGGCA 924 (SEQ ID NQ: 31) Lys⁴⁷→G1u⁴⁷ GCGGCGTCAGG G AGGACGACCGC925 (SEQ ID NO: 32) Arg⁵⁰→Cys⁵⁰ AGGAAGGACGAC T TGCACGGGCACCG 926 (SEQ IDNO: 33) Gly⁵²→Ser⁵² GACGACCGCACG A GCACCGGCACCCT 927 (SEQ ID NO: 34)Phe⁵⁹→Leu⁵⁹ ACCCTGTCGGTA C TCGGCATGCAGG  75 (SEQ ID NO: 35)Gln²¹⁴→Arg²¹⁴ TGCCAGCTGTACC G GAGATCGGGAGA  76 (SEQ ID NO: 36) PcDNA3CAAAAAGCTCC GC GGAGCTTGTATA 161 (SEQ ID NO: 37) (SmaI→KspI)*

EXAMPLE 20 Expression of TS Variants and Cytotoxicity Assay:Transfection of TS-Negative Cells with Wild-Type and Mutant Human TScDNA's

The mouse TS-negative FSthy21 cells were used as the host for DNAtransfections. This cell line, which is a TS-deficient derivative ofmouse FM3A cells, was routinely maintained in MEM medium supplementedwith 10% fetal bovine serum and 10×10⁻⁵ M thymidine. Transfections wereperformed by DOTAP, using 10 μg of DNA/culture. Four days aftertransfection, cells were placed in selective medium, which contain nothymidine and reduced folate. Colonies having the ability to grow in theabsence of thymidine were pooled and propagated as a mass culture.

EXAMPLE 21 Growth Inhibition Assay for Transfected Cells

Logarithmically growing suspension mouse TS-20 negative FSthy21 cellstransfected with wild-type or variant human TS cDNA were seeded in96-well plates at 1,000 cells/well in 180 μl of complete medium. Twohours later, drug (tomudex, AG337, BW1843U89 or FdUrd) was added and thecells were grown in drug-containing medium for an additional 7 days.Cell viability was measured by the alamar Blue™ assay. This methodincorporates an oxidation-reduction (REDOX) indicator that bothfluoresces and changes color in response to chemical reduction of growthmedium resulting from cell growth. To above 96-well cultured cells, 25μl of Alamar Blue (Alamar, Sacramento, Calif.) (10% of incubationvolume) were added according to the manufacturer's instructions. Theculture 96-well plates were then incubated at 37° C. for 4 hours. Viablecells induce chemical reduction of the media which results in a changein REDOX color from blue to red. The intensity of red color (andfluoresces) is proportional to the viable cells. After incubation,fluorescence is was read at 530-560 nm excitation wavelength and 590 nmemission wavelength by an automated plate reader (model EL340; Bio-Tek).Drug concentrations needed to reduce cell growth by 50% (IC₅₀ values)were determined graphically by plotting of cell growth verses inhibitorconcentration.

EXAMPLE 22 TS Purification and Characterization: Construction ofBacteria Expression Vectors with Human TS Variants

Human TS variants (I108A, F225W, G52S and D49G) selected for enzymekinetic characterization, were recloned into the protein expressionvector pET-17(b. DNA fragments covering the entire mutant human TS cDNAwere amplified using pcDNA3hTS* as the templates using two designedprimers. The 5′-primer contains a created NdeI restriction site and the3′-primer has XhoI site. After PCR amplification, the reaction mixturewas digested with the NdeI and XhoI enzymes and the desired DNA fragment(950 bp) was inserted to the corresponding sites of pET-17b vector. Thecorrect construction of pET-17bhTS* was verified by plasmid mapping andsequence analysis.

EXAMPLE 23 Expression of Recombinant Mutant Human TS and Preparation ofBacterial Crude Extract

The pET-17xbhTS* plasmid (wt or mutant TSs) was used to transform intoE. coli strain BL21 (DE3). Bacteria containing plasmids were grown at30° C. in 1 liter of tryptone phosphate medium [2% bacto-tryptone, 1.5%yeast extract, 0.2% sodium phosphate (dibasic), 0.1% potassium phosphate(monobasic), 0.8% sodium chloride and 0.2% glucose] supplemented with100 μg/ml of ampicillin (Moore et al., 1993). When the OD₆₀₀ reaches0.6-0.8, 1 mM of IPTG was added to induce synthesis of the humanprotein. After 5 hours of incubation, bacteria were harvested and thenwashed in TNE buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA).The harvested bacteria were resuspended in TNE buffer containing 20 mMBME and sonicated (10×30 sec, set 50) on ice. The debris was removed bycentrifugation at 37 000×g for 30 minutes at 4° C. to obtain the solubleextract. The supernatants were assayed for TS activity using thespectrophotometric assay and analyzed by SDS-PAGE. The expressed humanTS protein was visualized by staining with Coomassie Blue.

EXAMPLE 24 Purification of Wild-Type and Variants of Human TS

The wt and various mutant TSs were induced with 1 mM IPTG for 5 hours atwhich point the enzyme was induced to about 10%-15% of the solubleprotein. All of the purification procedures were performed at 0-4° C.and yielded protein of high purity for kinetic characterization. Step 1:Streptomycin Treatment. A 5% solution of streptomycin sulfate was addedthe extract (15 ml/100 ml). The suspension was stirred for 20 min. andthen centrifuged at 27 000×g for 30 min. Step 2: Ammonium Sulfate. Thesupernatant fraction from the previous step was brought to 30%saturation ammonium sulfate with solid ammonium sulfate, which wasstirred for 20 min. and centrifuged at 27 000×g for 30 min., and thepellet discarded. The resulting solution was then brought to 80%saturation and centrifuged as before. The precipitate was dissolved in10 ml of buffer (10 mM potassium phosphate, pH 7.4, 20 mM BME) anddialyzed overnight against the same buffer (2 liters). Step 3:Ion-Exchange Cellulose Chromatography. The dialyzed solution (100 to 200mg of protein) was loaded to 1.5×8-cm column of DE-52 equilibrated with10 mM KH₂PO₄, pH 7.4, 20 mM BME. The column was washed with the loadingbuffer at 1 ml/min. until no protein peaks were eluted and theconcentration of potassium phosphate changed to 25 mM. TS-containingfractions eluting with 50 mM KH₂PO₄, pH 7.4 20 mM BME were pooled andthen precipitated with solid ammonium sulfate to 80% saturation andcentrifuged at 27 000×g for 30 min. The ammonium sulfate precipitatewith human TS are stable stored at −20° C. Step 4: Phenyl-SepharoseCL-4B Chromatography. The pellet from the above step was dissolved in 20mM KH₂PO₄, pH 7.4 0.25 mM EDTA, 20 mM BME containing 0.8M ammoniumsulfate and loaded onto a 1.5×8-cm column of phenyl-Sepharose CL-4Bequilibrated with the same buffer. The column was washed with 200 ml ofthe loading buffer and TS-containing fractions eluted with 500 ml of adecreasing linear gradient of ammonium sulfate from 0.8 mM to 0.Fractions were concentrated by centrifugal ultrafiltration andprecipitated with solid ammonium sulfate and stored at −80° C. untiluse. TS purity was demonstrated by 12% SDS-PAGE.

EXAMPLE 25

Enzyme Assays for Wild-Type and Mutant Human TSs

TS activity was monitored spectrophotometrically at 340 nm as described(Wahba et al., 1961). Activity was determined at 30° C. The assaymixture contained 50 mM Tris.HCl, pH 7.4, 25 mM MgCl₂, 6.5 mMformadehyde, 1 mM EDTA, 75 mM BME and 100 μM dUMP, CH₂H₄folate (100 μM)was added to initiate the reaction. Black cuvettes lacked CH₂H₄folate. Aunit of activity is defined as the amount of enzyme required to convert1 μmol of dUMP to dTMP/minutes at 30° C. Protein was determined fromOD₂₈₀=0.87×10⁵ M⁻¹ cm⁻¹, which is equivalent to 1.43 OD₂₈₀/mg ofprotein.

EXAMPLE 26 K_(m) of CH₂H₄folate

Michaelis constants (K_(m)'s) for CH₂H₄folate and dUMP were determinedfrom initial velocity measurements, which were obtained by measuring thechange in OD₃₄₀ with a Shimadzu UV-2101 PC spectrophotometer. Fordetermination of K_(m) of CH₂H₄folate, the concentration of dUMP wasfixed at 200 μM while CH₂H₄folate was varied between 10 and 400 μM. Fordetermination of K_(m) of dUMP, CH₂H₄folate was present at aconcentration of 3 mM, and dUMP was varied between 1 to 300 μM.Steady-state kinetic parameters were subsequently obtained by anonlinear least-squares fit of the data to the Michaelis-Menten equationusing a computer program. The k_(cat)s were calculated by dividingV_(max) by the estimated concentration of enzyme used in the reaction.

EXAMPLE 27 K_(i) of Antifolates and FdUrd

Inhibition constants (K_(i)'s) were determined from the steady-stateinhibition reaction rates for mixtures of enzyme, dUMP, CH₂H₄folate andinhibitor. A high fixed CH₂H₄folate concentration (300 μM) and variableantifolate concentrations were used to measure the inhibition producedby tomudex, AG337 and BW1843U89 while a constant dUMP concentration andvaried FdUMP concentrations were applied for FdUMP.

EXAMPLE 28 AG337 in Human Sarcoma HT1080 Cells after Exposure toEthylmethanesulfonate

The development of drug resistance is a major limiting factor tosuccessful chemotherapy of cancer in humans. Cells in culture haveserved as an useful tool for the study of the mechanisms of drugresistance. Of the novel TS-targeted antifolates such as tomudex,BW1843U89 and AG337, tomudex has been intensively studied in culturedcell lines and several factors associated with resistance to tomudexhave been described, including decreased drug uptake, defectiveintracellular polyglutamylation as well as elevation of TS protein (Luet al., 1995). Mutations in the TS gene leading to expression of analtered enzyme with reduced affinity for tomudex, BW1843U89 or AG337 hasnot yet been reported.

The present invention isolated a mutant human TS which when transfectedinto bone marrow progenitor cells confers resistance to these TSspecific antifolates. In addition, the present invention evaluatedmechanisms of resistance to these novel TS inhibitors. In order toincrease the possibility of obtaining human TS mutations leading toantifolate resistance, human sarcoma HT1080 cells were exposed toethylmethanesulfonate (EMS), a monofunctional alkylating agent, followedby AG337 selection. Similar procedures were used to generate a number ofresistant clones to TMTX, a DHFR inhibitor. Both DHFR gene amplificationand mutations in DHFR were found (Fenin et al., 1993). The lipophilic TSinhibitor AG337 rather than tomudex or BW1843U89 was used as theselective drug since it does not utilize the reduced folate carries fortransport and is structurally precluded from polyglutamation, thusprobably narrowing the causes of drug resistance to TS geneamplification or TS mutations. Also, the development of drug-resistantsublines by random mutagenesis following single-step drug selectionmight increase the possibility of obtaining TS mutants that demonstratedecreased affinity for AG337 and other TS antifolate inhibitors.

A concentration of 400 μg of EMS/ml of culture medium was chosen forHT1080 cell exposure, which resulted in 80% inhibition of colonyformation. The IC₅₀ of AG337 for HT1080 cells is 2.1 μM, which wasobtained from drug cytotoxicity assay. A high concentration of 40 μM(19-fold of the IC₅₀) was used to select resistant clones based on thesolubility of this drug. Total forty-one AG337-resistant colonies wereobtained following EMS mutagenesis. In contrast, only 1 colony survivedfrom 1×10⁸ cells exposed to the same concentration of AG337 without EMSpretreatment. Thus, as expected, EMS exposure dramatically enhanced thefrequency of AG337-resistant colonies (10-fold). Stable resistantcolonies were continued in the presence of drug to expand to cell linesfor further analysis.

EXAMPLE 29 Single-Stranded Conformation Polymorphism Analysis ofResistant Cell Lines to Detect TS Mutants

For analysis of TS mRNA by the PCR-SSCP method, total cellular RNAs froma parental and 42 resistant cell lines (1 from the control experiment)expanded from individual colonies were transcribed into cDNA's usingreverse transcriptase. The single-stranded cDNA fragments thus obtainedwere amplified by the PCR to double-stranded DNA fragments (890 bp)using a set of two appropriate oligonucleotide primers (hTS-1A andhTS-3B), which have nucleotide sequences complementary to the codingregion (nt 50 to 69 and nt 939 to 915, respectively) of the TS mRNA.Human TS has unique 27 amino acids encoded by over 70% of G and C at theamino terminus, causing the problem for designing a specific bindingprimer much closer N-terminus than hTS-1A. Thus it is possible thatmutations in the first 70 nucleotides was missed. TS cDNA fragmentsobtained by RT-PCR were amplified again by 6 pairs of TS specificprimers to generate smaller fragments of DNA. The six regions of the TScDNA amplified are indicated in TABLE 3, the fragments with nucleotidelengths of 161 to 259 base pairs obtained being designated as A to F.These six regions, which overlapped each other and covered the mostcoding sequences of the TS cDNA of about 890 bp, were subjected to SSCPanalysis.

TABLE 3 DNA Fragments Amplified by Six-Pair of Primers for SSCP AnalysisRegion Primer Pair PCR Product Detected by SSC Region # hTS-1A/hTS-4B161 bp (nt 50-210) nt 70-187 (118 bp) A hTS-4A/hTS-1B 258 bp (nt 97-354)nt 118-333 (216 bp) B hTS-2A/hTS-5B 196 bp (nt 298-503) nt 320-480 (161bp) C hTS-5A/hTS-2B 195 bp (nt 451-645) nt 476-621 (146 bp) DhTS-3A/hTS-6B 229 bp (nt 594-822) nt 617-802 (186 bp) E hTS-6A/hTS-3B184 bp (nt 756-939) nt 780-914 (135 bp) F

After PCR amplification, DNA fragments were analyzed on SSCP gels. Theseparated single-strands DNA were visualized by either isotopic[(α³²P]dCTP labeling or ethidium bromide staining. Different runningtemperatures with or without 10% glycerol were tested to observeabnormally migrating SSCP bands to define optimal conditions. Theresults of DNA-SSCP analysis for TS cDNA are summarized on TABLE 4. Nineof 41 resistant cell lines (HT1080/1a, 2a, 1b, 2b, 1c, 2c, 1d, 2 d, and6e) showed single-stranded DNA fragments with mobility shifts inaddition to wildtype bands (see FIG. 2), indicating possible structuralchanges in those fragments due to mutations in the TS gene.

TABLE 4 DNA-SSCP Analysis of HT1080 and Resistant Cell Lines A B C D E FH1080 N³ N N N N N HT1080/A¹ N N N N N N HT1080/1a² N N N N N NHT1080/2a N N N N N N HT1080/1b B.S.⁴ B.S. N N B.S. B.S. HT1080/2b B.S.N N N N N HT1080/3b N N N N N N HT1080/4b N N N N N N HT1080/5b N N N NN N HT1080/6b N N N N N N HT1080/7b N N N N N N HT1080/1c B.S. N N N N NHT1080/2c B.S. N N N N N HT1080/3c N N N N N N HT1080/4c N N N N N NHT1080/5c N N N N N N HT1080/6c N N N N N N HT1080/7c N N N N N NHT1080/1d B.S. N N N N N HT1080/2d B.S. N N N N N HT1080/1e B.S. N N N NN HT1080/2e B.S. N B.S. N N B.S. HT1080/3e N N N N N N HT1080/4e N N N NN N HT1080/5e N N N N N N HT1080/6e N B.S. N N N N HT1080/7e N N N N N NHT1080/8e N N N N N N HT1080/9e N N N N N N HT1080/10e N N N N N NHT1080/11e N N N N N N HT1080/1f N N N N N N HT1080/2f N N N N N NHT1080/3f N N N N N N HT1080/4f N N N N N N HT1080/5f N N N N N NHT1080/6f N N N N N N HT1080/7f N N N N N N HT1080/8f N N N N N NHT1080/9f N N N N N N HT1080/10f N N N N N N HT1080/11f N N N N N NHT1080/12f N N N N N N ¹This cell line was obtained from the controlexperiment. The other 41 cell lines labeled by number plus a to f werefrom random mutagenesis experiment ²small a to f represent differentplates). ³N indicates that this fragment no band shifts. ⁴B.S. indicatesthat this DNA fragment had band shifts.

EXAMPLE 30 Tritium Release Assay for TS Activity in Whole Cells

The effect of AG337 on whole cell in situ TS activity was measured bythe tritium release assay in HT1080 cells and several resistant celllines with putative mutations. The cell lines HT1080/1b, HT1080/2b,HT1080/1c, HT1080/2c, HT1080/1d, HT1080/2d and HT1080/6e were resistantto AG337. At the minimal concentration of AG337 that resulted in nodetectable TS activity for HT1080, TS activity in the above mentionedresistant cell lines was not totally inhibited, showing those cells wereless sensitive to AG337 than the parental cells. This method provided arapid qualitative confirmation of whether or not a cell line isresistant to AG337.

EXAMPLE 31 Northern Blot Analysis

To examine whether single-step selection with relatively high AG337concentrations after EMS pretreatment led to changes in the expressionof the TS gene, cytoplasmic RNA from parental and the AG337-resistantcell line obtained without EMS exposure (HT1080/A) as well as theEMS-treated AG337-resistant cell lines with altered mobility on SSCPgels was analyzed by northern blot analysis using a human TS cDNA probe.The expression level of TS mRNA from the parental line (lane 1), andresistant cell lines (lanes 2-10) is shown in FIG. 3. Some AG337resistant sublines (HT1080/A, HT1080/2b, HT1080/2c, HT1080/2d andHT1080/2e) have an increase in TS mRNA, but there was no increase in TSmRNA in other AG337-resistant cell lines (HT1080/1b, HT1080/1c,HT1080/1d, HT1080/1e and HT1080/6e). The results suggested that someAG337-resistant cell lines with band shifts on SSCP gels derive theirresistance from a mechanism that does not appear to also involve theoverexpression of TS gene.

EXAMPLE 32 Western Blot Analysis

In order to provide further confirmation of the results obtained withNorthern blot analysis which showed some AG337-resistant cell lines didnot overexpress TS mRNA, expression of TS protein was measured bywestern blot analysis using a TS polyclonal antibody. FIG. 4 shows thatby comparison with the parental HT1080 cell line, some sublines(HT1080/1b, HT1080/1c, HT1080/1d and HT1080/1e) did not exhibitdetectable elevated levels of TS enzyme, and resistant sublines(HT1080/2b, HT1080/2c, HT1080/2d and HT1080/2e) expressed high levels ofTS protein, being consistent with the Northern blot results. However, ofinterest, the subline HT1080/6e had the same mRNA level as HT1080 butoverexpressed TS protein.

EXAMPLE 33 Analysis of DNA Fragments with Mobility Shifts on SSCP Gels

To elucidate the structural alterations that caused mobility shifts infragments detected by the PCR-SSCP analysis, sequencing of thesefragments was performed. Bands with mobility shifts and normal bandswere present on SSCP gels. Moreover, by comparison of the aberrantbands, the normal bands had a stronger signal in general. Thisobservation suggests that these cell lines contain both wild-type andmutant TS genes.

Direct isolation of DNA fragments from SSCP gels to perform PCRsequencing was attempted without success. It is possible that theoligonucleotides used, although, were suitable primers for PCRamplification, not useful for sequencing. DNA fragments carryingputative mutations on human TS gene were amplified and subsequentlysubcloned into the pCR-Script™ SK(+) vector. Several positive clones foreach sample were chosen and subjected to plasmid sequencing from bothdirections. Over one hundred clones containing independent fragmentswere screened by sequence analysis. Most sequencing results were foundto be identical to the wild-type human TS gene. However, twenty-fivepoint mutations were identified and are summarized in TABLE 5 which notincluded five silent mutations. None of mutations was identified inparental cells even when DNA fragments were sequenced more than 10times.

TABLE 5 Point Mutations in the TS Gene Identified by Sequence Analysisof DNA from Resistant Cell Lines Fragments Point Amino Acid ChangedAmino Acid Cell Lines Sequenced Mutation Changes Description HT1080/6e4A-1B AAG→GAG 47 Lys→Glu Always Lys or Arg HT1080/6e 4A-1B TAC→TGC 65Tyr→Cys Always Tyr or Phe HT1080/6e 4A-1B GTT→GCT 84 Val→Ala Always Valor Ile HT1080/6e 4A-1B CGC→TGC 50 Arg→Cys Conserved HT1080/6c 4A-1BGTG→ATG 79 Val→Met Conserved HT1080/1b 3A-3B GCC→ACC 228 Ala→ThrConserved HT1080/1b 3A-3B CAG→CGG 214 Gln→Arg Conserved HT1080/1b 3A-3BAAA→ATA 266 Lys→Ile Most is Lys HT1080/2b 1A-4B GAC→AAC 49 Asp→AsnConserved HT1080/2b 1A-4B ATC→ACC 40 Ile→Thr Always Ile or Val HT1080/1d1A-4B ACG→GCG 51 Thr→Ala Conserved HT1080/2d 1A-4B CGT→CAT 25 Arg→HisNot conserved HT1080/2d 1A-4B TTC→CTC 59 Phe→Leu Conserved HT1080/1e1A-4B GGC→AGC 52 Gly→Ser Always Gly or His HT1080/2e 4A-1B GAC→GGC 49Asp→Gly Conserved HT1080/2e 2A-2B GAC→GGC 130 Asp→Gly ConservedHT1080/2e 3A-3B AAA→TAA 266 Lys→Stop Most is Lys HT1080/2e 3A-3B ATT→ACT267 Ile→Thr Not conserved HT1080/2e 3A-3B ACG→ATG 234 Thr→Met ConservedHT1080/2e 3A-3B GAT→GGT 289 Asp→Gly Always Asp, Glu or Phe

Analysis of these nucleotide changes causing amino acid replacementsrevealed that 18 of 20 mutations were AT×GC or GC×AT transitions, and 2are transversions. In addition, 18 of 20 (or 18 of 25 including the 5silent mutations) resulted in amino acid changes in highly conservedresidues. In resistant HT1080/6e cells, for example, the arginine codon(CGC) at amino acid position 50 of the TS gene is mutated to a cysteinecodon (TGC) by a C to T transition. Arg⁵⁰ is a highly conserved residuewhich is believed to hydrogen bond with dUMP. In HT1080/1e cells, a G toA transition resulted in replacement of glycine (GGC) by serine (AGC) atamino acid residue 52 (see FIG. 5).

EXAMPLE 34 Mutagenesis of the Folate Binding Site of Human ThymidylateSynthase

Amongst the point mutations in the human TS gene of the sequencedisclosed in Genbank Accession number NM001071 (SEQ ID No. 38)identified by sequence analysis from random mutagenesis studies one atposition 50 is directly involved in substrate and cofactor binding. Thesubstitutions of amino acid residues located in binding regions maycause dramatic shifts of binding affinities to substrate. Previousstudies with DHFR have demonstrated that the positions 22, 31 and 34which interact with the PABA ring of H₂folate are hot spots formutagenesis and several point mutations on those sites leading to MTXresistance were found in drug resistant cell lines (Simonsen et al.,1983; Schweitzer et al., 1989). Crystal structures of TS have revealedthat Ile108, Leu221 and Phe225 are residues providing hydrophobicinteractions with the PABA moiety of folates when the ternary structureof TS bound nucleotide and folate is formed. None of these threeresidues, which all are very highly conserved, have been reported to bemutated previously in any TS species. Based on the above considerations,multiple substitutions were performed for Ile108, Leu221 and Phe225 inTS, including: (1) Trp, Ser, Leu or Tyr substitutions for Phe inposition 225; (2) Phe, Arg, Ala, Ile or Ser for Leu in position 221 and(3) Ala, Phe, Gly, Glu or Asn substitutions for Ile108.

The point mutations found by EMS pretreatment and following AG337selection include two (59 and 214) which are important for ligandbinding and structural stability, and six in the Arg50 loop whichbecomes more ordered by movement and reorientation upon ligand binding.Therefore, those eight mutations were chosen for further analysis, usingthe same procedures for the mutagenesis study of the three folatebinding sites. TABLE 6 shows that the mutated amino acid positions inhuman TS gene that are highly conserved and important in catalyticfunction and ligand binding.

TABLE 6 The Mutations in Human TS which are Highly Conservative forPrimary Structure and Important for Ligand Binding Positions in humanand cox L. casei thymidylate Sequence conservation Interactions withsynthase among 29 TS species CH₂H₄ folate or dUMP Phe225/ Highlyconserved with one Hydrophobic contact with Phe228 exception His PABA offolate Leu221/ Highly conserved with one Hydrophobic contact with Leu224exception Val PABA of folate Ile108/Ile81 Highly conserved with twoHydrophobic contact with exceptions Val and Tyr PABA of folateLys47/Lys20 Invariant in vertebrates Arg50 loop Asp49/ Strictlyinvariant Arg50 loop Asp22 Arg50/ Highly conserved with two Hydrogenbond with dUMP Arg23 exceptions Gly and C-terminus Thr51/Thr24 Highlyconserved with one Arg50 loop exception Gln Gly52/His25 Highly conservedwith five Arg50 loop exceptions; two His, Arg, Met & Pro Phe59/Phe32Highly conserved with two β-sheet i, forming part of exceptions Met andThr the substrate binding pocket Gln214/ Highly conserved with oneβ-sheet iii, a kick region for Gln217 exception Ala three β-sheetformation

EXAMPLE 35 Construction of Mutations in Human TS

The human TS expression vector pcDNA3hTS, containing the entire codingsequence of the human TS gene of the sequence disclosed in GenbankAccession number NM001071 (SEQ ID No. 38) with minor modifications ofthe N-terminal nucleotide codon, was constructed. The blunt-enddouble-stranded fragment of human TS cDNA (950 bp) generated bydigestion of the bacterial expression plasmid pET-17(bhTS with NdeI andHindIII restriction enzymes and following treatment with T4 DNApolymerase, was inserted to the unique EcoRV restriction site of vectorpcDNA3. The novel constructed plasmid pcDNA3hTS under control of the T7promoter was used to generate single strand DNA for site-directedmutagenesis and sequence analysis, as well as an mammalian expressionvector. Twenty-two human TS mutants, which included 8 mutations (K47E,D49N, D49G, R50C T51A, G52S, F59L and Q214R) identified from randommutagenesis studies and another twelve multiple replacements of Ile108,Leu221 and Phe225 were prepared by site-directed mutagenesis usingpcDNA3hTS as a template, following the instructions described by theTransformer™ site-directed mutagenesis kit (2nd version, Clontech). Theselective primer was designed to change the unique restriction site SmaIto KspI and twenty-two mutagenic primers with 1 to 3 nucleotide changeswere optimized with a computer program for oligo design. Sequencing thecoding regions demonstrated that the expected substitution had beenintroduced and that no other alteration had occurred (data not shown).TABLE 9 lists the site of the mutations obtained in the eukaryoticexpression plasmids.

TABLE 7 Eukaryotic Expression Plasmids Containing Mutants of Human TSand the Results of Rescue Experiments Mouse TS-Negative Cells a.a FM3ATS cells num- Reason for Eukaryotic Protein expression surviving byReference ber Point mutation a.a change change expression systemtransfection number wt pcDNA3hTS161* pET-17xbhTS161 yes 161 225 TTC→TGGPhe→Trp S. information pcDNA3hTS162 pET-17xbhTS162 yes 162 225 TTC→TCCPhe→Ser S. information pcDNA3hTS163 no 163 225 TTC→CTC Phe→Leu S.information pcDNA3hTS83 yes 83 225 TTC→GCC Phe→Tyr S. informationpcDNA3hTS84 yes 84 221 CTC→TTC Leu→Phe S. information pcDNA3hTS164 no164 221 CTC→CGC Leu→Arg S. information pcDNA3hTS165 no 165 221 CTC→GCCLeu→Ala S. information pcDNA3hTS85 yes 85 221 CTC→ATC Leu→Ile S.information pcDNA3hTS86 yes 86 221 CTC→AGC Leu→Ser S. informationpcDNA3hTS87 yes 87 108 ATC→GGC Ile→Ala S. information pcDNA3hTS175pET-17xbhTS175 yes 175 108 ATC→TTC Ile→Phe S. information pcDNA3hTS176yes 176 108 ATC→GGC Ile→Gly S. information pcDNA3hTS270 no 270 108ATC→GAG Ile→Glu S. information pcDNA3hTS271 no 271 108 ATC→AAC Ile→AsnS. information pcDNA3hTS272 no 272 47 AAG→GAG Lys→Glu EMS treatmentpcDNA3hTS925 yes 925 49 GAC→AAC Asp→Asn EMS treatment pcDNA3hTS923 no923 49 GAC→GGC Asp→Gly EMS treatment pcDNA3hTS928 pET-17xbhTS928 yes 92850 CGC→TGC Arg→Cys EMS treatment pcDNA3hTS926 no 926 51 ACG→GCG Thr→AlaEMS treatment pcDNA3hTS924 no 924 52 GGC→AGC Gly→Ser EMS treatmentpcDNA3hTS927 pET-17xbhTS927 yes 927 59 TTC→CTC Phe→Leu EMS treatmentpcDNA3hTS75 no 75 214 CAG→CGG Gln→Arg EMS treatment pcDNA3hTS76 no 76*the number indicates different mutations and is consistent withreference number.

EXAMPLE 36 Rescue of TS-negative Cells by Transfection of Human TScDNA's

The stable transfection of human TS variants into a TS-negative mousecell was used to demonstrate whether or not the altered TS protein hadenough catalytic activity for allowing normal growth in the absence ofthymidine. Mouse TS-negative cells (FSthy21) were transfected with aplasmid (pcDNA3hTS*) encoding wild-type or a various mutant human TSenzyme by standard DOTAP transfection procedures. The host cells aredeficient in TS, which are unable to survive without exogenousthymidine. The results of the rescue experiment showed that 11transfectants of TS variants (I108A, I108F, F225W, F225L, F225Y, L221A,L221I, L221S, K47E, D49G, and G52S) as well as wild-type TS were able tocomplement growth of TS-negative cells in selective medium lackingthymidine (TABLE 7). In contrast, cells were unable to survive withtransfection of other 11 human TS variants. For each survivingtransfectant, at least 12 individual clones were isolated by soft agaror limiting dilution in 96-well plates. Those clones were expanded tocell lines by growth in normal media. Three cell clones for eachtransfectant were randomly selected for drug sensitivity assay.

EXAMPLE 37 Growth Sensitivity to Antifolates and FdUrd

Before determining growth sensitivity to three antifolates (tomudex,AG337, and BW1843U89) and 5-fluoro-2′-deoxyuridine (FdUrd), the levelsof human TS protein for the transfectants were demonstrated by westernblot analyses. Two I108A mutant clones (175-1 and 175-2) expressedlevels of TS protein at the same level as that of a control clone(161-2) transfected with the wild-type vector (TABLE 8-11). Using the TSprotein level expressed by the above three cell clones as a standard,cell clone 175-3 had slightly higher and clones 161-1 and 161-2 hadlower levels of protein. For the other 10 transfectants with human TSvariants, cell clones expressing similar TS levels as the standard werechosen.

The effect of three antifolates and FdUrd on the growth characteristicsof transfectants that stably express various TS proteins was evaluatedby cytotoxicity studies. Cell growth was measured by the alamar blueassay. IC₅₀ values were calculated as the concentration of inhibitorrequired to inhibit growth by 50% compared to wild-type transfectedcells grown under identical conditions. For various transfectants, theIC₅₀ values of antifolates (AG337, tomudex, and BW1843U89) and FdUrd arepresented in TABLES 8-11, and the graphical comparisons of the IC₅₀values are given in FIGS. 6-9. As indicated, statistically significantdifferences in the IC₅₀ values were obtained: (1) I108A transfectantsdisplay resistance to tomudex and AG337 with IC₅₀ values at least 43-and 76-fold greater than wild-type transfectants, respectively; (2) D49Gand G52S transfectants confer resistance to AG337 (40- and 12-foldrespectively); (3) F225W transfectants are 22-fold resistant toBW1843U89; (4) G52S, D49G and F225W mutants demonstrate FdUrd 97-, 26,and 25-fold resistance.

TABLE 8 Tomudex (ZD1694) Sensitivity in TS-negative Cells Transfected byWild Type and Various Mutant Human TS cDNA's Transfected TS-negativeIC₅₀ values Ratio of IC₅₀ values Reference cells and its clone (×10⁻⁹M)¹ for mutant/wt cells number wild-type/clone (1) 2.37 ± 0.18 161-1wild-type/clone (2) 1.80 ± 0.47 161-2 wild-type/clone (3) 3.37 ± 0.34161-3 1108A/clone (1) 97.9 ± 17.1    54(2)² 175-1 1108A/clone (2) 76.8 ±7.3    43(2) 175-2 1108A/clone (3) 174 ± 7    97(2) 175-3 1108F/clone(2) 1.52 ± 0.20 0.84(2) 176-2 F225W/clone (2) 1.76 ± 0.01 0.98(2) 162-2F225L/clone (3) 1.33 ± 0.29 0.74(2)  83-3 F225Y/clone (1) 1.53 ± 0.170.85(2)  84-1 L221A/clone (2) 1.46 ± 0.09 0.81(2)  85-2 L221I/clone (1)1.98 ± 0.40  1.1(2)  86-1 L221S/clone (2) 1.80 ± 0.06  1.0(2)  87-2K47E/clone (2) 1.53 ± 0.04 0.85(2) 925-2 D49G/clone (1) 1.76 ± 0.090.98(2) 928-1 G52S/clone (3) 1.94 ± 0.05  1.1(2) 927-3 ¹IC₅₀ values wereobtained following 7-day exposures from full dose-response curves andrepresent the mean ± SE (standard error) of at least 2 separateexperiments involving duplicate samples from replicate cultures. ²Thenumber in parentheses indicated which clone of wild-type TS transfectedcells was compared.

TABLE 9 AG337 Sensitivity Expressed in TS-negative Cells Transfected byWild Type and Various Mutant Human TS cDNA's Transfected TS-negativeIC₅₀ values Ratio of IC₅₀ values Reference cells and its clone (×10⁻⁷M)¹ for mutant/wt cells number wild-type/clone (1) 1.64 ± 0.08 161-1wild-type/clone (2) 3.83 ± 0.69 161-2 wild-type/clone (3) 1.49 ± 0.21161-3 1108A/clone (1) 305 ± 37    80(2)² 175-1 1108A/clone (2) 290 ± 25  76(2) 175-2 1108A/clone (3) 766 ± 28  200(2)  175-3 1108F/clone (2)11.4 ± 1.2  7.0(2) 176-2 F225W/clone (2) 0.70 ± 0.35 1.0(1) 162-2F225L/clone (3) 1.64 ± 0.56 1.0(1)  83-3 F225Y/clone (1) 2.38 ± 0.451.5(1)  84-1 L221A/clone (2) 8.96 ± 0.34 5.5(1)  85-2 L221I/clone (1)8.51 ± 0.18 5.2(1)  86-1 L221S/clone (2) 7.38 ± 0.28 4.5(1)  87-2K47E/clone (2) 8.25 ± 0.61 5.0(1) 925-2 D49G/clone (1) 66.7 ± 2.5  40(1) 928-1 G52S/clone (3) 19.1 ± 0.71  12(1) 927-3 ¹IC₅₀ values wereobtained following 7-day exposures from full dose-response curves andrepresent the mean ± SE (standard error) of at least 2 separateexperiments involving duplicate samples from replicate cultures. ²Thenumber in parentheses indicated which clone of wild-type TS transfectedcells was compared.

TABLE 10 BW1843U89 Sensitivity in TS-negative Cells Transfected by WildType and Various Mutant Human TS cDNA's Transfected TS-negative IC₅₀values Ratio of IC₅₀ values Reference cells and its clone (×10⁻⁹ M)¹ formutant/wt cells number wild-type/clone (1) 2.78 ± 0.12 161-1wild-type/clone (2) 3.68 ± 0.14 161-2 wild-type/clone (3) 1.44 ± 0.28161-3 1108A/clone (1) 22.8 ± 4.2   6.2(2)² 175-1 1108A/clone (2) 27.2 ±3.1  7.4(2) 175-2 1108A/clone (3) 28.8 ± 1.3  7.8(2) 175-3 1108F/clone(2) 3.44 ± 0.03 1.2(1) 176-2 F225W/clone (2) 61.1 ± 10.3  22(1) 162-2F225L/clone (3) 7.84 ± 0.44 2.8(1)  83-3 F225Y/clone (1) 7.32 ± 0.172.6(1)  84-1 L221A/clone (2) 7.41 ± 0.25 2.7(1)  85-2 L221I/clone (1)7.53 ± 0.25 2.7(1)  86-1 L221S/clone (2) 7.63 ± 0.26 2.7(1)  87-2K47E/clone (2) 7.55 ± 0.23 2.7(1) 925-2 D49G/clone (1) 14.7 ± 1.2 5.3(1) 928-1 G52S/clone (3)  8.4 ± 0.7  3.0(1) 927-3 ¹IC₅₀ values wereobtained following 7-day exposures from full dose-response curves andrepresent the mean ± SE (standard error) of at least 2 separateexperiments involving duplicate samples from replicate cultures. ²Thenumber in parentheses indicated which clone of wild-type TS transfectedcells was compared.

TABLE 11 5-Fluoro-2′-deoxyuridine (FdUrd) Sensitivity in TS-negativeCells Transfected by Wild Type and Various Mutant Human TS cDNA'sTransfected TS-negative IC₅₀ values Ratio of IC₅₀ values Reference cellsand its clone (×10⁻¹¹ M)¹ for mutant/wt cells number wild-type/clone (1)2.90 ± 0.03 161-1 wild-type/clone (2) 8.96 ± 0.62 161-2 wild-type/clone(3) 2.86 ± 0.02 161-3 1108A/clone (1) 140 ± 19   16(2)² 175-11108A/clone (2) 105 ± 18  12(2) 175-2 1108A/clone (3) 137 ± 25  15(2)175-3 1108F/clone (2) 17.6 ± 2.8  6.1(1)  176-2 F225W/clone (2) 73.8 ±2.6  25(1) 162-2 F225L/clone (3) 16.5 ± 2.8  5.7(1)   83-3 F225Y/clone(1) 3.01 ± 0.06 1.0(1)   84-1 L221A/clone (2) 11.6 ± 3.1  4.0(1)   85-2L221I/clone (1) 38.1 ± 4.1  13(1)  86-1 L221S/clone (2) 3.49 ± 0.551.2(1)   87-2 K47E/clone (2) 16.4 ± 4.5  5.7(1)  925-2 D49G/clone (1)76.3 ± 1.9  26(1) 928-1 G52S/clone (3) 282 ± 28  97(1) 927-3 ¹IC₅₀values were obtained following 7-day exposures from full dose-responsecurves and represent the mean ± SE (standard error) of at least 2separate experiments involving duplicate samples from replicatecultures. ²The number in parentheses indicated which clone of wild-typeTS transfected cells was compared.

To determining if different expressed TS levels in transfectantscontributed to drug sensitivity changes of the IC₅₀ values of fourdrugs, multiple clones of wild-type and I108A mutant transfections werecompared to TS levels. Clone 175-3, having the highest TS levels, ismore resistant to tomudex and AG337 than clone 175-1 and 175-2 but therewas no marked increase in IC₅₀ values to BW1843U89 and FdUrd. Thebiggest difference in IC₅₀ values between clones 175-2 and 175-3 forAG337 was less than 3-fold. Similar results with relatively less changesof drug sensitivity were also obtained using three individual clones ofwild-type transfectants. These data suggested that the changes in drugsensitivity by different clones transfected with the same vector areroughly proportional to their expressed protein levels. However, themajor reason causing dramatic changes in IC₅₀ values was due toexpression of TS variants and the levels of TS protein was lessimportant.

EXAMPLE 38 Subcloning and Expression of Human TS in E. coli

Based on the drug sensitivity results presented, human TS mutationsI108A (175), F225W (162), D49G (928) and G52 (927) were selected forenzyme kinetic studies. The fragments encompassing the entire human TScDNA with different mutations, amplified by a pair of designed primersthat contained NdeI and XhoI restriction sites in the 5′ and 3′ primersrespectively, were subcloned into the pET-17×b vector utilizingcorresponding NdeI and XhoI sites. The pET system was developed toprovide the high yields of soluble protein in E. coli. These proteinexpression vectors were designated as pET-17×bhTS175, pET-17×bhTS162,pET-17×bhTS928, and pET-17×bhTS927 (see Table 7). The expression ofwild-type and mutant TSs was carried in a derivative of the E. colistrain BL21 after IPTG induction. The activity of the enzyme wasmonitored and found to be highest at 4-5 h after the addition of IPTG.In the absence of IPTG the activity from extracts of transformed BL21cells was much lower and probably represents background E. coli TSactivity. E. coli extracts after 4-5 h of IPTG induction were analyzedby SDS-PAGE and revealed an intensely staining band at a molecular massof about 36 kDa, absent from extracts of the host E. coli cells. Thisnew protein band was estimated to represent about 10-20% of the totalsoluble protein in the extract. The crude extracts from bacteria cellstransformed by mutant TS vectors had similar high levels of productionof the altered proteins.

EXAMPLE 39 Purification of Mutant TS Proteins

A purification procedure using sequential ion exchanger/phenyl-sepharosechromatography was adopted for purifying wild-type and mutant human TSproteins (Ciesla et al., 1995). The procedure of Ciesla et al. wasmodified in that the human TS was eluted from phenyl-sepharose using alinear gradient of ammonium sulfate of 0.6 M to 0 instead of 0.8 to 0.4M employed for rat proteins. After purification, a single majorcomponent on SDS-PAGE gel migrating with an apparent molecular weight ofhuman TS protein was observed for all mutant enzymes. Purity wasestimated to be higher than 90% as determined by densitometric scanning.

EXAMPLE 40 Kinetic Properties of Mutant Enzymes

In order to obtain more detailed information about the catalytic andligand-binding properties of these TS variants, kinetic parameters suchas V_(max) and K_(m)'s for substrate and cofactor, and K_(i)'s forinhibitors were evaluated. The results for wild-type and mutant TSs arepresented in TABLE 12.

The Michaelis constant (K_(m)) values for dUMP was not significantlydifferent between the wild-type and F225W mutant forms while thecatalytic efficiency (k_(cat)) of F225W was even higher than the k_(cat)of wild-type TS. However, K_(m) values for CH₂H₄folate differed markedlywith a 4-fold increase for F225W mutant over wild-type. More dramaticchanges were noted with the I108A mutant. Its affinity for cofactor wasdecreased to a much larger degrees (22-fold) than for dUMP (4-fold). TheK_(m) for CH₂H₄folate and dUMP of the D49G variant was increased 4-foldover wild-type TS. Both I108A and D49G mutants showed diminishedcatalytic activity with k_(cat) values 3-fold lower than wild-type TS.

TABLE 12 Kinetic Parameters for Wild-Type and Mutant Human ThymidylateSynthases K_(m) (CH₂H₄folate) Km (dUMP) k_(cat) Enzyme (μM) (μM) (sec⁻¹)Reference #^(b) wt 32.4 4.4 0.68 161 F225W 165 3.6 2.3 162 I108A 746 150.22 175 G52S ND^(a) ND ND 927 D49G 122 12 0.16 928 ^(a)not determined,^(b)refers to Table 7.

TABLE 13 Binding affinity of Tomudex, AG337, BW1843U89 and FdUMP forWild-Type and Mutant Human Thymidylate Synthase Inhibition ConstantsVariants of TS (K_(i) (nM) wt 1108A F225W G52S D49G Tomudex 7.0  4100 87 ND* 53 Variant/wt —  580 13 ND 7.6 BW1843U89 0.09^(b)  2400 23 ND 180Variant/wt 27000^(c) 260 ND 2000 AG337 16  1400 5.3 ND 130 Variant/wt  91 0.35 ND 8.1 FdUMP 11   22 25 ND 23 Variant/wt   2.0 2.3 ND 2.1Reference #^(d) 161  175 162 927 928 ^(a)not determined; ^(b)obtainedfrom previous report; ^(c)by comparison with previous data; ^(d)refersto Table 7

EXAMPLE 41 Transfection of Wild Type (TS161) and Mutant (TS175) Plasmid

DNA into Mouse Bone Marrow Progenitor Cells Twenty micrograms of plasmidDNA (TS161 or TS175) and ten micrograms of DOTAP were diluted to 100 μlwith HBS buffer and incubated at room temperature for 10 minutes. Themixture was added to 2×10⁶ mouse bone marrow cells suspended in 0.5 mlof IMDM medium (in a 35 mm culture dish). After a 4 hour incubation at37° C., 2 ml of IMDM with 30% FBS was added to each dish and theincubation was continued for 24 hours. At this time, another 4 ml offresh medium with 10% FBS was added to each dish and incubationcontinued for an additional 48 hours.

Transfected cells prepared as described above were distributed into10×35 mm dishes (3×10⁵ cells in 6 ml IMDM media/dish). The mediacontained 1% methyl cellulose, 20% FBS, 10% WEHI-3B conditioned media,1% sodium pyruvate, 1 mM mercaptoethanol, 100 μg/ml of penicillin, 100μg/ml streptomycin, 1% essential amino acids, 1.5% nonessential aminoacids and 0.5% ascorbate. Drug was added at the indicated concentrationto each dish and the cells were cultured at 37° C. (5% CO₂) for 10-14days. CFU-GM colonies (>50 cells) were counted and larger colonies(>1000 cells) HPPCFU-C (high proliferation colony forming cells) werecounted under the microscope. TABLE 14 shows that mouse marrow CFU-CMcolonies and HPPCFU colonies transfected with a plasmid containing themutant TS (TS175) had greater resistance to tomudex (D1694) and AG337than mouse marrow CFU-CM colonies and HPPCFU colonies transfected with aplasmid containing the wild type TS (TS161).

TABLE 14 Resistance to tomudex (D1694) and AG337 in mouse marrow CFU- CMcolonies and HPPCFU colonies after transfection with plasmids containingwild type TS (TS161) and mutant TS (TS175) CFU-GM Assay tomudex AG337 02 × 10⁻⁸ M 1 × 10⁻⁸ M 2 × 10⁻⁷ M 1 × 10⁻⁸ M N.B.M.  2  0 14(17%)  010(12%) TS161 108  0 28(26%)  0 20(19%) TS175 144 14(10%) 64(44%) 12(8%)60(42%) HPPCFU-C Assay tomudex AG337 1 × 10⁻⁸ M 1 × 10⁻⁷ M N.B.M.  0  0TS161  0  0 TS175  9(6%)  8(6%)

Discussion

Several point mutations in E. coli and L. casei TS gene were previouslymade by cassette and site-directed mutagenesis. The procedures includedtwo steps; generated mutants were first screened for their catalyticactivity by complementing the growth of TS-negative E. coli cells in theabsence of thymine and kinetic characterizations were subsequentlyperformed for functional mutants. Alternative approaches were adoptedfor the studies focusing on human TS mutants. Firstly, randommutagenesis were performed by exposure of human sarcoma HT1080 cells toan alkylating agent (EMS) and selection with AG337, to generate putativemutations leading to AG337 resistance. These isolated mutants wereutilized to measure their binding to other TS inhibitors such astomudex, BW1843U89 and FdUrd. Moreover, mutations made by EMS couldoccur anywhere in the entire TS gene and were not limited to specificgene regions as in cassette mutagenesis. However, as it is almostimpossible to obtain double or triple point mutations in a nucleotidecodon by random mutagenesis, therefore some desirable amino acidsubstitutions are excluded by this approach. The functional roles ofindividual amino acid residues were examined, especially their effectson the binding of the folate cofactor or inhibitors by generatingspecific mutations. Fourteen mutations at positions 108, 221 and 225that are involved in hydrophobic interactions of TS with cofactor orinhibitors were created by site-directed mutagenesis. The targetresidues were chosen because they are highly conserved and because theyare important for folate binding as indicated by X-ray crystallographicstudies. In addition, multiple substitution studies for these threeamino acid residues have not yet been reported on all species of TS.Mouse TS-deficient cells instead of TS-negative E. coli cells were usedas a host for expression of mutant TS enzymes, which allowed anassessment of catalytic activity and provided a mammalian test systemfor evaluating the effects of inhibitors of TS.

The present invention obtained human TS variants that conferredresistance to novel antifolates, with a minimal changes in the catalyticactivity of the enzyme. Such variants are of interest for severalreasons, particularly for their use in gene therapy to protecthemotopoietic progenitors from drugs, such as tomudex. The resultsdescribed herein demonstrate that several human TS mutants havedesirable properties that support their use in such gene therapystudies. The present invention obtained information on the mode ofbinding of CH₂H₄folate and inhibitors to TS indicating that thedifferent TS inhibitors bind to TS in different ways.

Human TS Mutants Identified in EMS-Exposed Cells

Single-stranded conformation polymorphism (SSCP) is a simple andsensitive approach to analyze nucleotide changes which result in alteredmobility of DNA fragments on non-denaturing polyacrylamide gels.However, SSCP sensitivity varies dramatically with the size of the DNAfragment. The optimal size of DNA fragments are approximately 150 bp inlength. When the presence of point mutations in EMS-exposedAG337-resistant cells was investigated by SSCP analysis, 6-pairs ofprimers were used to span the cDNA for TS, each amplified 150-260 bpfragments. By screening of almost the entire coding sequence of human TSgene from AG337-resistant HT1080 sublines, shifted bands in addition tonormal migrating bands were observed on SSCP gels, indicating thepresence of wild-type and mutant TS genes in the sample. Polymorphismscan be detected when mutant DNA comprised as little as 3% of the totalgene copies in a PCR mixture (Hongyo et al., 1993). However, SSCP cannotdiscriminate between pre-existing mutations and those mutationsintroduced by Taq polymerase errors during amplification.

After EMS exposure, a relatively large number of drug resistant clonescould be obtained by selection with TMTX, a lipophilic DHFR inhibitor.An increase in frequency of drug resistant clones was also observed inthe mutagenesis experiment using AG337 as a selective drug.Unexpectedly, more than 20 mutations of human TS were identified inAG337-resistant sublines. In order to explain the large number ofmutations, it was postulated that EMS exposure and Taq polymerase errorscontributed to the point mutations found after SSCP and sequenceanalysis. Other less likely possibilities that would cause mutationsinclude selection with AG337 which could act as a weak mutagen, andinfidelity of AMV reverse transcriptase.

For mutations caused by EMS exposure as well as Taq polymerase errors,transitions are more likely to predominant over transversions. For Taqpolymerase, the most common transition was AT×GC (72% of all changes),and the remaining base substitutions were observed at roughly equivalentfrequencies (Tindall et al., 1988). However, in contrast to infidelityof Taq polymerase, EMS-induced GC×AT transitions were much more commonthan AT×GC transitions (65% of all).

Based on above information, if all or most of the mutations identifiedin EMS-exposed cells were caused by Taq polymerase error, AT×GCtransitions should be dominant. Otherwise, some mutations could begenerated by EMS exposure. The mutation results showed that AT×GC andGC×AT transitions were roughly equal (see TABLE 6), indicating that someor most of those mutations were generated by EMS exposure. Thisconclusion is supported by evidence that none of the mutations weredetected in controls, in which parent HT1080 cells without EMS and AG337exposure were tested. Most amino acid substitutions were found in highlyconserved positions, suggesting that these variants were likely to leadto drug resistance. Therefore, 8 mutations were selected for studies inmouse TS-negative cells for directly examining the ability of thesemutations to allow growth in the absence of thymidine. Cytotoxicitystudies showed that D49G and G52S TS variants display resistance to theselective drug AG337, providing additional proof that these mutationswere involved in the AG337 resistant phenotype selection.

The Arg50 Loop and Drug Resistance

Surprisingly, of the 20 point mutations identified from the randommutagenesis, six occurred in the highly conserved Arg50 loop. This loopconnects elements of protein secondary structure, an α-helix A (residues30-43) near the N-terminus and a β-sheet i (residues 54-66) (Montfort etal., 1990). When Agouron designed folate analogues such as AG337, thequinazoline ring system was kept intact, based on observations from thehumanized E. coli TS model (Reich et al., 1992). Hydrogen-bonding waspredicted to occur between the carboxylate of Asp218 and hydrogen on N-3of quinazoline ring, and another between N-1 of quinazoline ring and afixed H₂O molecule which in turn is hydrogen bonded to Val313 and Arg50.Therefore, Arg50 is believed to play a in the structure-based drugdesign. Moreover, in the native unbound TS, the Arg50 loop is mobile.Once the ternary complex is formed, the carboxylate of the C-terminalresidue forms a hydrogen bond network with Arg50, which shifts 0.5 Å tointeract with the phosphate of dUMP and hydrogen-bond with N-1 ofCH₂H₄folate via H₂O. This flexible residue Arg50 seems to be a bridge tolink the enzyme C-terminus, substrate and cofactor (or antifolates)together. The movement of the Arg50 residue is accompanied by adjustmentand reorientation of its neighbor residues, indicating that the entireArg50 loop undergoes relocation and experiences new interactions. Forexample, the hydrophobic atoms of Thr51 has contacts with the buriedVal313 side chain after movement. Besides structure studies, theresidues in Arg50 loop have been studied by mutagenesis of E. coli andL. casei TS. The present invention indicates that the modification ofthe loop Arg50 causes changes on binding affinity leading to drugresistance, which is related to not only folate but also nucleotides.

Arg50 is extremely sensitive to substitution by other amino acids, asshown with both L. casei and E. coli TS. The R50C mutant of human TS iscatalytically inactive, and the T51A and D49N mutants are also nottolerated, which is consistent with comparably altered L. casei and E.coli TS. However, 3 other point mutations (K47E, D49G and G52S) retainTS catalytic function. Cytotoxicity assays also showed that expressedD49S and G52S mutant protein in mouse TS-negative cells confersresistance to AG337 with IC₅₀ values 40- and 12-fold greater than cellsexpressing wild-type TS expressed cells respectively. These mutanttransfected cell lines also display resistance to FdUrd (26- and 97-foldrespectively) but not to tomudex or BW1843U89. The K_(m) values forCH₂H₄folate and dUMP of the D49S mutant protein also demonstrated thatthe mutation equally affects both the substrate and cofactor bindingwith about a 4-fold increase in K_(m) values. By comparison withwild-type, the K47E mutant did not show a difference in binding to thesefour drugs, which might be related to its position being relatively farfrom Arg50. Based on the above results and structural information of theArg50 loop, the basis for reduced AG337 and FdUMP binding with D49S andG52S mutants may result from impaired movement of the Arg50 loop,especially for the Arg50 residue which is involved in folate and dUMPbinding. More marked changes by some mutations such as R50C, D49N andT51A result in inactive TS enzymes, while small structural perturbationof Arg50 loop due to these mutations may compensated for by the localadjustment of neighbor residues, which still maintain contacts withligands in the new position.

Ile108 Mutants and the Role of Ile108

The human TS active site is comprised of a hydrophobic region thatincludes Trp109, Asn112, Met311, Ile108, Leu221 and Phe225. Multiplesubstitutions on positions 108, 221 and 225 were performed bysite-directed mutagenesis, because these changes could alter theconformation of the TS active site and or the electrostatic environment,resulting in altered binding affinities with substrate and inhibitorsleading to drug resistance.

Although Ile108 is a highly conserved amino acid residue, it showsflexibility of its side chain when antifolates occupy the folate bindingsite. When human TS lipophilic inhibitors such as AG337 were designed bymolecular modeling, it was predicted that the side chain of Ile108 wouldmove toward the distal phenyl ring of AG337 to make the desired nonpolarinteraction, resulting in the calculated minimum-energy conformation.This movement and modeled interaction were subsequently observed in thecrystal structure of TS complexed to AG337. The shift of the Ile108 sidechain upon AG337 binding suggested that the spatial relationship ofIle108 to bound folate and inhibitors such as AG337 is little differentdue to the changed molecular structure of inhibitor. Some mutations onposition 108 may affect the binding of cofactor and inhibitors (AG337,tomudex and BW1843U89) that can occupy the cofactor binding site. Thefirst two mutations made were Phe and Ala substitutions. Because ofsmaller side chain of alanine than isoleucine, the movement of sidechain of Ala may not be enough to contact the phenyl ring of AG337. Lossor weakening of these interactions in the variants must also reduce thebinding energy, resulting in dramatic changes of affinity of AG337 toTS. For phenylalanine, a non-polar amino acid, containing a phenyl groupthat has less flexibility, it was expected that if the stacking ofphenyl-phenyl rings between inhibitor and altered human TS is impeded,TS will adopt a new conformation by reorientation of amino acidresidues, thus causing a large change in enzyme behavior.

The results of cytotoxicity assays were that Ile108 replacement byresidues with a smaller side chain (Ala) weakened binding of AG337 andtomudex, with respective IC₅₀ values at least 78- and 43-fold greaterthan value obtained by wild-type human TS. In contrast, the increase ofthe IC₅₀ value of BW1843U89 was relatively less (6-fold), which mayreflect that the large molecular structure of BW1843U89 retains contactwith the side chain of alanine. Moreover, since Ile108 is not one ofresidues of the substrate binding pocket, I108A mutants did not affectthe FdUrd binding much with only a 12-fold increase in the IC₅₀ value.

In contrast, little effects on catalysis and inhibitor binding wereobserved for the I108F mutant. This variant behaved as same as wild-typeTS, suggesting that the side hydrophobic chain substitution did notchange the spatial interaction between enzyme, cofactor or inhibitors.In order to further study the role of Ile108 on cofactor and antifolatebinding, this residue was replaced by Asn (a polar a.a.), Glu (an acidica. a.) and Gly (a non-polar a.a. without a side chain). None of thesethree mutant enzymes could complement mouse TS-deficient cells in theabsence of thymidine, showing those changes greatly decreased thecatalytic efficiency due to the loss of hydrophobic contacts.

These mutagenesis studies showed that Ile108 could toleratesubstitutions with hydrophobic residues (Phe and Ala) but not Gly, Asnand Glu. Moreover, the drastic differences in folate binding andcatalytic activity among these variants indicate that position 108 isquite sensitive to mutagenesis and plays a critical role in folatebinding. These results strongly suggest that the I108A mutation wasresponsible for the altered properties of human TS and was sufficient toconfer significant AG337 and tomudex resistance, because it showeddistinctive effects on binding affinity among three antifolates anddifferent properties on catalysis compared to the other four mutants.

Some decrease in catalytic efficiency due to reduced affinity ofsubstrate and cofactor binding was also observed with the I108A mutant.However, I108A was able to allow mouse TS-negative cells to grow in theabsence of thymidine and to result in resistance to tomudex and AG337.In addition, transfection of mouse marrow cells with the I108A mutantcDNA resulted in more tomudex-resistant colony survival for the I108Amutant expressed cells as compared to mouse marrow cells transfectedwith wild-type human TS, indicating I108A mutant is a good candidate forgene transfer studies to protect bone marrow from tomudex or AG337toxicity.

Phe225 Mutants and the Role of Phe225

Phe225 was targeted for site-directed mutagenesis, because based on thecrystal structure of TS, this position contributes the major hydrophobicforce on binding of the PABA moiety of CH₂H₄folate, and exhibited alarge shift upon ternary complex formation. For accommodating differentligands, Phe225 underwent dramatic side chain movement. For example, theorientation of the phenyl group of Phe225 toward CB3717 and H₂folate ischanged on binding to these ligands. The aromatic ring of Phe225 stacksagainst the propargyl group of CB3717, which could be important for thestability of CB3717 binding to TS. However, the Phe225 side chain movesaway when H₂folate instead of CB3717 binds causing more weak andexpansive van der Waals contacts, which may allow H₂folate to dissociatefrom TS. These observations indicate that when TS forms a complex withCH₂H₄folate, H₂folate, CB3717, tomudex or AG337, which all have a pterinor quinazoline ring, the mobile phenyl group of Phe225 has enough spaceto accommodate these ligands by different hydrophobic interactions andit is not necessary to form aromatic-aromatic non-polar interaction byface-to-face interactions between Phe225 and the folate molecule.BW1843U89 binds differently, as a consequence of an expandedbenzoquinaline ring, and results in reorientation of the Phe225 phenylring (turn 90°) to form an aromatic π stacking with the isoindolinylring of BW1843U89.

The interactions of Phe225 with cofactor and various antifolates appearto be quite different. Phe225 was therefore replaced by tryptophan,tyrosine, leucine or serine. The results of transfection of mouseTS-negative cells demonstrated that this highly conserved residue couldbe substituted by Trp, Leu and Tyr but not by Ser. Also, the F225S andF225L mutant enzymes did not show drastic changes in binding of threeantifolates (AG337, tomudex, BW1843U89) and FdUrd. In contrast, theF225W variant conferred resistance to BW1843U89 and FdUrd, withincreased IC₅₀ values of 22- and 25-fold respectively. But, it did notlead to cross-resistance to AG337 and tomudex.

The above results indicated that the replacement of Phe225 withtryptophan, tyrosine, leucine or serine had very different effects onenzyme activity and binding affinity of inhibitors. The F225S mutantexhibited severely diminished TS catalytic activity, presumably becausethe hydroxyl side chain of serine decreased the hydrophobic interactionbetween TS and the PABA moiety of folate. Substitutions with tyrosine, aaromatic amino acid also containing a hydroxyl moiety, retained TSfunction. The aromatic group of Tyr may contribute hydrophobic contactsto folate compounds, along with negative binding factors caused by thehydroxyl polar group. The F225W mutant but not the F225L mutantconferred resistance to BW1843U89. The loss of the hydrophobicinteraction (aromatic π stacking) between the tryptophan side chain andthe isoindolinyl ring of BW1843U89 may account for the lost of bindingenergy for BW1843U89.

Mutations of Position 221

In the crystal structure of the human TS, Leu221, an almost invariantresidue in all reported TS sequences, was a residue involved inhydrophobic contacts with bound cofactor or inhibitors. Five pointmutations (phenylalanine, leucine, isoleucine, serine or arginine) wereintroduced at position 221 of the human TS via site-directed mutagenesisin order to evaluate whether these mutations would change the ligandbinding properties of the enzyme.

Mouse TS-negative cells were rescued by transfection with Ala, Ile andSer variants but not Phe and Arg variants. The presence of an Arg, apositive charged residue, substitution for Leu221 causing TS functionloss, is explained by the loss of hydrophobic interactions. Forphenylalanine substitution, a hydrophobic amino acid, because of itsbulk (Phe, 142 Å³ and Leu, 107 Å³), may result in the L221F variantbeing inactive. However, when the volume of non-polar side chain wasunchanged (for Ile substitution) or became smaller (for Ala) and anhydroxyl group added (for Ser), these mutations did not cause a largechange in enzyme behavior. The L221A, L221I and L221S variants of humanTS also did not show big differences on inhibitor binding affinities. Itmay suggest that the steric position of Leu221, unlike Phe225 andIle108, is hardly moved or reoriented when cofactor or inhibitors havingdifferent molecular structures bind to enzyme.

Random Mutagenesis

Random mutagenesis by EMS exposure and following AG337 selectionresulted in the generation of a large number of resistant clones.Without EMS pretreatment, only 1 ({fraction (1/10)}⁸ vs 4¼×10⁸) clonesurvived the selecting dose of AG337. Fourty one of these clones wereexpanded to obtain stable AG337-resistant cell lines. DNA-SSCP analysissuggested that 9 of 41 AG337-resistant cell lines with altered mobilityon SSCP gels may have acquired mutations in the TS gene. Analysis ofthese 9 AG337-resistant sublines by whole cell in situ TS assay as wellas Northern and Western blotting revealed that some resistant sublinesdemonstrated elevation of TS mRNA and enzyme levels, andcross-resistance to other TS-directed drugs tomudex and FdUrd, but somesublines did not overexpress TS mRNA and protein. Twenty mutations in TSin AG337-resistant cell lines were identified, resulting in geneamplification as well as mutations in the coding region. Of 8 mutationschosen for rescue studies of mouse TS-negative cells, three TS mutants(K47E, D49G and G52S) were able to allow growth of TS-negative cells inthe absence of thymidine, indicating they retain TS catalytic activity.D49S or G52S transfected in mouse TS-negative cells confer resistance toAG337 with IC₅₀ values 40- and 12-fold greater than wild-type TStransfected cells respectively. They also display resistance to FdUrd(respective 26- and 97-fold) but not to tomudex or BW1843U89. Thestructural perturbation of the Arg50 loop due to D49S or G52S mutationsmay cause resistance to AG337 and FdUrd. The K_(m) for CH₂H₄folate anddUMP of the D49G variant was increased 4-fold over wild-type TS.

Site-Directed Mutagenesis

Three amino acids, determined to be important for hydrophobicinteractions between the folate cofactor (inhibitors) and human TS, werechosen for site-directed mutagenesis studies. Ile108 was mutated to Ala,Phe, Gly, Glu and Asn. Only I108A and I108F TS mutants were functional.I108A mutant transfected cells confer resistance to tomudex and AG337with respective IC₅₀ values at least 43- and 78-fold greater thanwild-type TS transfected cells but not to BW1843U89 and FdUrd. The I108Amutant also was found to have a decrease in k_(cat) and an increase inK_(m) (CH₂H₄folate). The changes in the side chain due to I108A mutationmay cause the loss of hydrophobic interactions between position 108 andthe phenyl ring of AG337 and tomudex, resulting in reduced bindingaffinity of these molecules. Secondly, Phe225 was replaced by Trp, Ser,Leu and Tyr. F225W, F225L and F225Y are active enzymes. The F225W mutantdisplayed resistance to BW1843U89 without changes in V_(max) and K_(m)(dUMP). The perturbation of the aromatic π stacking between thetryptophan side chain and the isoindolinyl ring of BW1843U89 may accountfor the lost binding energy for BW1843U89 to TS. Thirdly, L221A, L221I,L221S, L221F and L221R mutants were created by site-directedmutagenesis. TS catalytic activity could be restored by Ala, Ile and Serbut not Phe and Arg mutants. Fourthly, I108A (for tomudex and AG337),G52S (for 5-FU) and F225W (for BW1843U89) human TS mutants arerepresentative examples of mutants for gene transfer studies.

The present invention isolated and characterized human TS mutantsconferring antifolates or 5-PU resistance. Based on the presentedfindings, there are two major applications including resistant genetransfer studies and crystal structure analysis of TS variants. Forexample, resistance to 5-FU has been related to insufficient inhibitionof tumor TS. Higher 5-FU doses may increase the success of chemotherapyof certain cancer. The transduction of hematopoietic precursor cellswith the G52S mutant TS cDNA would allow dose-intense therapy of 5-FU incancer patient by decreasing myelotoxicity. Another importantapplication is X-ray crystallographic studies of three mutants (G52S,I108A and F225W). The three-dimensional structural data would provideinformation of how the mutated residues participate in inhibitor bindingor how the altered residues interfere with inhibitor binding throughsteric hindrance (for F225W), the loss of hydrophobic interaction (forI108A) or structural perturbation (for G52S). The knowledge of thedifference of wild-type and mutant human TS structures may also be ofvalue in the design of new TS inhibitors with desirable properties.

The following tables 15-17 summarize additional proof that these mutantsconfer resistance to chemotherapeutic agents by increasing the number ofdrug resistant CFU-GM colonies formed by murine bone marrow cellsinfected with retroviral vectors containing these mutant TS cDNAs.

TABLE 15 Colony Forming Unit-Granulocyte Macrophage (CFU-GM) assayincubated with 5-FluoroUracil for 10 days No drug 10⁻⁵ M 5-FU 10⁻⁶ M5-FU CFU-C* HPPCFU-C** CFU-C CFU-C HPPCFU-C Normal Bone Marrow 160 400    16(10%) 0   Wt TS 168 44 0    91(54%)  6(14%) G52S mut-TS 162 5310(6%) 117(72%) 21(40%) *CFU-C: colony forming units; **HPPCFU-C: highproliferative potential CFU-C

TABLE 16 incubated with Tomudex ^(R) (D-1694) No drug 2 × 10⁻⁸ M 1 ×10⁻⁸ M CFU-C* HPPCFU-C** CFU-C CFU-C HPPCFU-C Normal Bone Marrow 160 400   16(10%) 0   Wt TS 168 44 10(6%)  33(20%) 10(23%) I108A mut-TS 148 4925(15%) 53(36%) 26(53%) *CFU-C: colony forming units; **HPPCFU-C: highproliferative potential CFU-C

TABLE 17 incubated with AG337 (Thymitaq) No drug 5 × 10⁻⁷ M 1 × 10⁻⁷ MCFU-C* HPPCFU-C** CFU-C CFU-C HPPCFU-C Normal Bone Marrow 160 40 0   16(10%) 0   Wt TS 168 44 0    71(42%) 13(30%) I108A mut-TS 148 49 9(6%)110(36%) 38(78%) *CFU-C: colony forming units; **HPPCFU-C: highproliferative potential CFU-C

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Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

39 1 15 DNA artificial sequence Nucleotide sequence in the 5′ codingregion of human recombinants cDNA of thymidylate synthase (TS) gene inpET-17(bhTS) vector. 1 atgcctgtgg ccggc 15 2 15 DNA artificial sequenceAltered nucleotide sequence in the 5′ coding region of humanrecombinants cDNA of thymidylate synthase (TS) gene in pET-17(bhTS)vector. 2 atgcttgttg ctggt 15 3 20 DNA artificial sequence primer_bind50..69 Sense primer hTS-1A for PCR amplification of part of 890 basepair fragment of human TS gene from nucleotide 50 to the C-terminus 3cacaggagcg ggacgccgag 20 4 21 DNA artificial sequence primer_bind354..334 Antisense primer hTS-1B for PCR amplification of part of 890base pair fragment of human TS gene from nucleotide 50 to the C-terminus4 caaaaagtct cgggatccat t 21 5 22 DNA artificial sequence primer_bind298..319 Sense primer hTS-2A for PCR amplification of part of 890 basepair fragment of human TS gene from nucleotide 50 to the C-terminus 5gagctgtctt ccaagggagt ga 22 6 24 DNA artificial sequence primer_bind645..622 Antisense primer hTS-2B for PCR amplification of part of 890base pair fragment of human TS gene from nucleotide 50 to the C-terminus6 tctctggtac agctggcagg acag 24 7 26 DNA artificial sequence primer_bind594..616 Sense primer hTS-3A for PCR amplification of part of 890 basepair fragment of human TS gene from nucleotide 50 to the C-terminus 7ctgccagttc tatgtggtga acagtg 26 8 24 DNA artificial sequence primer_bind939..915 Antisense primer hTS-3B for PCR amplification of part of 890base pair fragment of human TS gene from nucleotide 50 to the C-terminus8 aacagccatt tccattttaa tagt 24 9 21 DNA artificial sequence primer_bind97..117 Sense primer hTS-4A for PCR amplification of part of 890 basepair fragment of human TS gene from nucleotide 50 to the C-terminus 9tacctggggc agatccaaca c 21 10 23 DNA artificial sequence primer_bind210..188 Antisense primer hTS-4B for PCR amplification of part of 890base pair fragment of human TS gene from nucleotide 50 to the C-terminus10 ttcatctctc aggctgtagc gcg 23 11 25 DNA artificial sequenceprimer_bind 451..475 Sense primer hTS-5A for PCR amplification of partof 890 base pair fragment of human TS gene from nucleotide 50 to theC-terminus 11 tcagattatt caggacaggg agttg 25 12 23 DNA artificialsequence primer_bind 503..481 Antisense primer hTS-5B for PCRamplification of part of 890 base pair fragment of human TS gene fromnucleotide 50 to the C-terminus 12 atggtgtcaa tcactctttg cag 23 13 24DNA artificial sequence primer_bind 756..779 Sense primer hTS-6A for PCRamplification of part of 890 base pair fragment of human TS gene fromnucleotide 50 to the C-terminus 13 gggagatgca catatttacc tgaa 24 14 20DNA artificial sequence primer_bind 822..803 Antisense primer hTS-6B forPCR amplification of part of 890 base pair fragment of human TS genefrom nucleotide 50 to the C-terminus 14 tctgggttct cgctgaagct 20 15 24DNA artificial sequence primer_bind Mutagenic oligonucleotide sequenceused to obtain Phe225 to Trp225 point mutation in hTS 15 cggtgtgccttggaacatcg ccag 24 16 24 DNA artificial sequence primer_bind Mutagenicoligonucleotide sequence used to obtain Phe225 to Ser225 point mutationin hTS 16 cggtgtgcct tccaacatcg ccag 24 17 24 DNA artificial sequenceprimer_bind Mutagenic oligonucleotide sequence used to obtain Phe225 toLeu225 point mutation in hTS 17 ctcggtgtgc ctctcaacat cgcc 24 18 24 DNAartificial sequence primer_bind Mutagenic oligonucleotide sequence usedto obtain Phe225 to Tyr225 point mutation in hTS 18 cggtgtgccttacaacatcg ccag 24 19 25 DNA artificial sequence primer_bind Mutagenicoligonucleotide sequence used to obtain Leu221 to Phe221 point mutationin hTS 19 ggagacatgg gcttcggtgt gcctt 25 20 26 DNA artificial sequenceprimer_bind Mutagenic oligonucleotide sequence used to obtain Leu221 toArg221 point mutation in hTS 20 gagacatggg ccgcggtgtg cctttc 26 21 24DNA artificial sequence primer_bind Mutagenic oligonucleotide sequenceused to obtain Leu221 to Ala221 point mutation in hTS 21 gagacatgggcgccggtgtg cctt 24 22 22 DNA artificial sequence primer_bind Mutagenicoligonucleotide sequence used to obtain Leu221 to Ile221 point mutationin hTS 22 gagacatggg catcggtgtg cc 22 23 24 DNA artificial sequenceprimer_bind Mutagenic oligonucleotide sequence used to obtain Leu221 toSer221 point mutation in hTS 23 gagacatggg cagcggtgtg cctt 24 24 22 DNAartificial sequence primer_bind Mutagenic oligonucleotide sequence usedto obtain Ile108 to Ala108 point mutation in hTS 24 gggagtgaaagcctgggatg cc 22 25 26 DNA artificial sequence primer_bind Mutagenicoligonucleotide sequence used to obtain Ile108 to Phe108 point mutationin hTS 25 caagggagtg aaattctggg atgcca 26 26 22 DNA artificial sequenceprimer_bind Mutagenic oligonucleotide sequence used to obtain Ile108 toGly108 point mutation in hTS 26 gggagtgaaa ggctgggatg cc 22 27 22 DNAartificial sequence primer_bind Mutagenic oligonucleotide sequence usedto obtain Ile108 to Glu108 point mutation in hTS 27 gggagtgaaagagtgggatg cc 22 28 22 DNA artificial sequence primer_bind Mutagenicoligonucleotide sequence used to obtain Ile108 to Asn108 point mutationin hTS 28 gggagtgaaa aactgggatg cc 22 29 25 DNA artificial sequenceprimer_bind Mutagenic oligonucleotide sequence used to obtain Asp49 toAsn49 point mutation in hTS 29 gtcaggaagg acaaccgcac gggca 25 30 25 DNAartificial sequence primer_bind Mutagenic oligonucleotide sequence usedto obtain Asp49 to Gly49 point mutation in hTS 30 tcaggaagga cggccgcacgggcac 25 31 25 DNA artificial sequence primer_bind Mutagenicoligonucleotide sequence used to obtain Thr51 to Ala51 point mutation inhTS 31 aaggacgacc gcgcgggcac cggca 25 32 23 DNA artificial sequenceprimer_bind Mutagenic oligonucleotide sequence used to obtain Lys47 toGlu47 point mutation in hTS 32 gcggcgtcag ggaggacgac cgc 23 33 25 DNAartificial sequence primer_bind Mutagenic oligonucleotide sequence usedto obtain Arg50 to Cys50 point mutation in hTS 33 aggaaggacg actgcacgggcaccg 25 34 26 DNA artificial sequence primer_bind Mutagenicoligonucleotide sequence used to obtain Gly52 to Ser52 point mutation inhTS 34 gacgaccgca cgagcaccgg caccct 26 35 25 DNA artificial sequenceprimer_bind Mutagenic oligonucleotide sequence used to obtain Phe59 toLeu59 point mutation in hTS 35 accctgtcgg tactcggcat gcagg 25 36 26 DNAartificial sequence primer_bind Mutagenic oligonucleotide sequence usedto obtain Gln214 to Arg214 point mutation in hTS 36 tgccagctgtaccggagatc gggaga 26 37 25 DNA artificial sequence primer_bind Selectionprimer for destroying the unique SmaI restriction site to generateanother unique KspI site on pcDNA3vector 37 caaaaagctc cgcggagctt gtata25 38 1536 DNA Homo sapiens Wild type human thymidylate synthase cDNA(Genbank Accession number IM 001071) 38 gggggggggg ggaccacttg gcctgcctccgtcccgccgc gccacttggc ctgcctccgt 60 cccgccgcgc cacttcgcct gcctccgtcccccgcccgcc gcgccatgcc tgtggccggc 120 tcggagctgc cgcgccggcc cttgccccccgccgcacagg agcgggacgc cgagccgcgt 180 ccgccgcacg gggagctgca gtacctggggcagatccaac acatcctccg ctgcggcgtc 240 aggaaggacg accgcacggg caccggcaccctgtcggtat tcggcatgca ggcgcgctac 300 agcctgagag atgaattccc tctgctgacaaccaaacgtg tgttctggaa gggtgttttg 360 gaggagttgc tgtggtttat caagggatccacaaatgcta aagagctgtc ttccaaggga 420 gtgaaaatct gggatgccaa tggatcccgagactttttgg acagcctggg attctccacc 480 agagaagaag gggacttggg cccagtttatggcttccagt ggaggcattt tggggcagaa 540 tacagagata tggaatcaga ttattcaggacagggagttg accaactgca aagagtgatt 600 gacaccatca aaaccaaccc tgacgacagaagaatcatca tgtgcgcttg gaatccaaga 660 gatcttcctc tgatggcgct gcctccatgccatgccctct gccagttcta tgtggtgaac 720 agtgagctgt cctgccagct gtaccagagatcgggagaca tgggcctcgg tgtgcctttc 780 aacatcgcca gctacgccct gctcacgtacatgattgcgc acatcacggg cctgaagcca 840 ggtgacttta tacacacttt gggagatgcacatatttacc tgaatcacat cgagccactg 900 aaaattcagc ttcagcgaga acccagacctttcccaaagc tcaggattct tcgaaaagtt 960 gagaaaattg atgacttcaa agctgaagactttcagattg aagggtacaa tccgcatcca 1020 actattaaaa tggaaatggc tgtttagggtgctttcaaag gagcttgaag gatattgtca 1080 gtctttaggg gttgggctgg atgccgaggtaaaagttctt tttgctctaa aagaaaaagg 1140 aactaggtca aaaatctgtc cgtgacctatcagttattaa tttttaagga tgttgccact 1200 ggcaaatgta actgtgccag ttctttccataataaaaggc tttgagttaa ctcactgagg 1260 gtatctgaca atgctgaggt tatgaacaaagtgaggagaa tgaaatgtat gtgctcttag 1320 caaaaacatg tatgtgcatt tcaatcccacgtacttataa agaaggttgg tgaatttcac 1380 aagctatttt tggaatattt ttagaatattttaagaattt cacaagctat tccctcaaat 1440 ctgagggagc tgagtaacac catcgatcatgatgtagagt gtggttatga actttatagt 1500 tgttttatat gttgctataa taaagaagtgttctgc 1536 39 313 PRT Homo sapiens Wild type human thymidylate synthaseamino acid sequence (Genbank Accession number NP001062) 39 Met Pro ValAla Gly Ser Glu Leu Pro Arg Arg Pro Leu Pro Pro 5 10 15 Ala Ala Gln GluArg Asp Ala Glu Pro Arg Pro Pro His Gly Glu 20 25 30 Leu Gln Tyr Leu GlyGln Ile Gln His Ile Leu Arg Cys Gly Val 35 40 45 Arg Lys Asp Asp Arg ThrGly Thr Gly Thr Leu Ser Val Phe Gly 50 55 60 Met Gln Ala Arg Tyr Ser LeuArg Asp Glu Phe Pro Leu Leu Thr 65 70 75 Thr Lys Arg Val Phe Trp Lys GlyVal Leu Glu Glu Leu Leu Trp 80 85 90 Phe Ile Lys Gly Ser Thr Asn Ala LysGlu Leu Ser Ser Lys Gly 95 100 105 Val Lys Ile Trp Asp Ala Asn Gly SerArg Asp Phe Leu Asp Ser 110 115 120 Leu Gly Phe Ser Thr Arg Glu Glu GlyAsp Leu Gly Pro Val Tyr 125 130 135 Gly Phe Gln Trp Arg His Phe Gly AlaGlu Tyr Arg Asp Met Glu 140 145 150 Ser Asp Tyr Ser Gly Gln Gly Val AspGln Leu Gln Arg Val Ile 155 160 165 Asp Thr Ile Lys Thr Asn Pro Asp AspArg Arg Ile Ile Met Cys 170 175 180 Ala Trp Asn Pro Arg Asp Leu Pro LeuMet Ala Leu Pro Pro Cys 185 190 195 His Ala Leu Cys Gln Phe Tyr Val ValAsn Ser Glu Leu Ser Cys 200 205 210 Gln Leu Tyr Gln Arg Ser Gly Asp MetGly Leu Gly Val Pro Phe 215 220 225 Asn Ile Ala Ser Tyr Ala Leu Leu ThrTyr Met Ile Ala His Ile 230 235 240 Thr Gly Leu Lys Pro Gly Asp Phe IleHis Thr Leu Gly Asp Ala 245 250 255 His Ile Tyr Leu Asn His Ile Glu ProLeu Lys Ile Gln Leu Gln 260 265 270 Arg Glu Pro Arg Pro Phe Pro Lys LeuArg Ile Leu Arg Lys Val 275 280 285 Gly Lys Ile Asp Asp Phe Lys Ala GluAsp Phe Gln Ile Glu Gly 290 295 300 Tyr Asn Pro His Pro Thr Ile Lys MetGlu Met Ala Val 305 310

What is claimed is:
 1. A mutated human thymidylate synthase (TS), saidmutated synthase differing from wild type TS having the amino acidsequence of SEQ ID No. 39 at amino acid residue 49, amino acid residue52, amino acid residue 108, amino acid residue 221 or amino acid residue225.
 2. The mutated human TS of claim 1, wherein said amino acid residue49 is mutated to an amino acid selected from the group consisting ofglycine and asparagine.
 3. The mutated human TS of claim 1, wherein saidamino acid residue 52 is mutated to serine.
 4. The mutated human TS ofclaim 1, wherein said amino acid residue 108 is mutated to an amino acidselected from the group consisting of alanine, phenyalanine, glycine,glutamic acid and asparagine.
 5. The mutated human TS of claim 1,wherein said amino acid residue 221 is mutated to an amino acid selectedfrom the group consisting of phenyalanine, arginine, alanine, isoleucineand serine.
 6. The mutated human TS of claim 1, wherein said amino acidresidue 225 is mutated to an amino acid selected from the groupconsisting of tryptophan, serine, leucine and tyrosine.
 7. A cDNAderived from a wild type cDNA, said wild type cDNA having the nucleotidesequence of SEQ ID No. 38, wherein said derived cDNA has been mutated toencode the mutated human TS of claim
 1. 8. A DNA vector, said vectorcomprising: DNA derived from a wild type cDNA, said wild type cDNAhaving the nucleotide sequence of SEQ ID No. 38, wherein said DNA hasbeen mutated to encode the mutated human TS of claim
 1. 9. A host cell,said host cell transfected with the DNA vector of claim 8, wherein saidhost cell produces the mutated human TS of claim
 1. 10. The host cell ofclaim 9, wherein said cell is a mammalian hematopoietic cell.
 11. Thehost cell of claim 10, wherein said cell is a peripheral blood stemcell.